Pharmaceutical compositions and methods

ABSTRACT

This invention relates the use of cortisol blockers (e.g., glucocorticoid receptor [GR] antagonists) for the treating or preventing viral infections, treating or preventing treatment resistant prostate cancer, treating or preventing neoplasia, and treating or preventing infection related to acute or chronic injury or disease.

PHARMACEUTICAL COMPOSITIONS AND METHODS

This application is a Divisional application of U.S. Ser. No.15/825,982, filed Nov. 29, 2017, which is a Divisional application ofU.S. Ser. No. 15/370,680 filed Dec. 6, 2016 now U.S. Pat. No. 9,855,284issued Jan. 2, 2018, which is a Divisional application of U.S. Ser. No.15/222,062, filed Jul. 28, 2016, now U.S. Pat. No. 9,598,459, issuedMar. 21, 2017, which claims benefit under 35 U.S.C. § 119(e) of U.S.Provisional Patent Application No. 62/282,525, filed Aug. 3, 2015; U.S.Provisional Patent Application No. 62/220,583, filed Sep. 18, 2015; U.S.Provisional Patent Application No. 62/241,875, filed Oct. 15, 2015; U.S.Provisional Patent Application No. 62/271,038, filed Dec. 22, 2015; U.S.Provisional Patent Application No. 62/280,073, filed Jan. 18, 2016; U.S.Provisional Patent Application No. 62/296,673, filed Feb. 18, 2016; U.S.Provisional Patent Application No. 62/299,120, filed Feb. 24, 2016; U.S.Provisional Patent Application No. 62/314,046, filed Mar. 28, 2016; U.S.Provisional Patent Application No. 62/339,909, filed May 22, 2016; U.S.Provisional Patent Application No. 62/339,906, filed May 22, 2016; U.S.Provisional Patent Application No. 62/342,103, filed May 26, 2016; U.S.Provisional Patent Application No. 62/342,965, filed May 29, 2016; U.S.Provisional Patent Application No. 62/342,966, filed May 29, 2016; U.S.Provisional Patent Application No. 62/342,967, filed May 29, 2016; U.S.Provisional Patent Application No. 62/345,319, filed Jun. 3, 2016; U.S.Provisional Patent Application No. 62/345,095, filed Jun. 3, 2016; U.S.Provisional Patent Application No. 62/349,235, filed Jun. 13, 2016; U.S.Provisional Patent Application No. 62/352,624, filed Jun. 21, 2016; U.S.Provisional Patent Application No. 62/352,611, filed Jun. 21, 2016; U.S.Provisional Patent Application No. 62/352,572, filed Jun. 21, 2016; U.S.Provisional Patent Application No. 62/352,576, filed Jun. 21, 2016; U.S.Provisional Patent Application No. 62/358,211, filed Jul. 5, 2016; U.S.Provisional Patent Application No. 62/361,125, filed Jul. 12, 2016, thedisclosures of which are incorporated herein in their entirety.

BACKGROUND OF THE INVENTION

This invention relates the use of cortisol blockers (e.g.,glucocorticoid receptor [GR] antagonists) for the treating or preventingviral infections, treatment resistant prostate cancer, treating orpreventing neoplasia, and treating or preventing infection related toacute or chronic injury or disease.

Rapid advances in technology of all kinds and advances in travel andglobalization have had substantial impacts on improving the humancondition within the United States and internationally. However, bothall of these advances have proven to be a double-edged sword, allowingfor the easy spread of invasive species and disease, whether it beaccidental or intentional. The United States government has beenproactive in its work to legislate and fund medical countermeasures workin response to the potential for public health emergencies initiated bythe introduction of pathogens. Key among these responses have been the2004 Project Bioshield Act and the 2006 Pandemic and All HazardsPreparedness Act, the latter of which provides opportunities through theBiomedical Advanced Research and Development Authority (BARDA).

The National Institute of Allergy and Infectious Diseases Institutes ofHealth (NIAID), a component of the National Institute of Health (NIH),maintains a list of emerging infectious diseases and pathogens forpurposes of prioritization and research guidance. Pathogens areprioritized from A-C based on the traits of transmissibility, morbidity,mortality and diagnostics. Additionally, a list of emerging pathogensand diseases is included, which are unclassified with a priority level.These lists were used as a springboard for study of a series ofcompounds developed by Palisades Therapeutics (PT), a division of PopTest Oncology LLC, that have been demonstrated to have antiviralactivity against a wide range of human pathogens.

The following sections will provide detailed information on thecompounds PT150 and PT155, and viral pathogens from the NIAID listsagainst which they show, or are hypothesized to show, activity. Weconfirmed this activity both in vitro and in animal models. Thisactivity includes Zika virus. We believe their activity levels asantivirals against Flavivirus, and possibly other RNA virus, prioritizestheir movement into clinical testing.

PT-150 is a re-purposed drug acquired from a major pharmaceuticalcompany that has a transferable IND that would allow it to be rapidlyplaced into a human clinical trial population if it is determined thatthis is justified. The compound previously completed all of its INDenabling preclinical studies, a significant Phase 1 study in humans andtwo large Phase 2 human trials for psychotic depression. PT-150 hasunique properties as an antiviral drug in that it has the ability topenetrate to sanctuary sites like the brain, thymus, and testicles. It,thus, may have the potential to clear virus from these sites throughdirect antiviral activity whereby infected cells are inhibited fromreplicating virus and are cleared by apoptosis.

PT-155 is a derivative of PT-150 that has demonstrated even higheractivity related to the putative mechanism of action that could resultin even greater efficacy. PT150 would require a Phase III ClinicalTrial. PT155 would require IND enabling safety pharmacology andtoxicology studies and IND enabling CMC programs.

PT-150 (formerly Org34517) and its derivative molecules PT-155 andPT-156 have at least two general mechanisms of anti-viral action effectagainst a broad array of viruses infecting animals and humans. The firstanti-viral effects are mediated through binding of these molecules toglucocorticoid response elements (GREs) present in some viral genomes.The second is through binding of these molecules to phosphatidylserine(PS) present in the envelope of all enveloped viruses.

The mechanism of action through binding to GRE's is as follows:

Viruses that infect animals and humans infect cells by placing theirgenetic material within the cytoplasm and/or nucleoplasm of the infectedcell. “Response elements” within the genome, which may comprise codingregions or non-coding regions, respond to molecular signaling of thehost cell and/or other elements of the virus' own molecular network.Viruses often have GREs, namely response elements that are under theinfluence of glucocorticoid signaling mediated by the binding ofcortisol (or other glucocorticoids) to the glucocorticoid receptor(GCR).

The viruses that have been identified as having GRE's include: HepatitisC virus, Bovine Viral Diarrhea virus, Ebola-like viruses, Hepatitis Bvirus, Mouse mammary tumor virus, Human Immunodeficiency Virus-1(HIV-1), Varicella-Zoster virus (chicken pox; VZV), Cytomegalovirus(CMV), Human Herpes Virus-6 (HHV-6), Human Herpes Virus-7 (HHV-7),Kaposi's Sarcoma-Associated Herpes virus (or Human Herpes Virus-8;HHV-8), Variola (Small Pox) virus, Vaccinia virus, Cowpox virus,Monkeypox virus

Binding of PT-155, as a GCR antagonist, as well as of its derivatives,including but not limited to PT-155 and PT-156, also modulates the viralGRE to directly or indirectly inhibit fundamental viral functions(including, but not limited to genetic replication, production ofvirus-associated proteins, assembly of genetic material and viralproteins into complete viruses, increasing genetic diversity, promotionof viral active or passive virus release from the cell, and viralinfectivity).

Moreover, these viruses contain viral DNA transcripts that arepro-viruses that enable viruses to remain latent in sequestered cellularcompartments in the body; these DNA pro-viral genomes are responsiblefor latent infection which may erupt into full viral replication insituations when an individual becomes immunocompromised or whensuppressive anti-viral regimens are interrupted. Binding of thesemolecules inactivates such pro-viral activities by either inactiving thepro-virus directly or by causing pro-viral genomic mutations thattrigger p53 mediated host cell apoptosis. Either way, the pro-viralgenome is destroyed. In these susceptible viruses, PT-150, PT-155, andPT-156, as well as other possible derivatives of these molecules, willlead to cure of chronic viral infection.

The mechanisms of anti-viral action related to PS binding are asfollows:

PS is normally sequestered to the inner leaflet of the plasma membranebilayer, but during apoptosis the mechanism that normally maintains PSin the inner leaflet is down-regulated, allowing the appearance of PS onthe cell surface. PS exposure is recognition signal for phagocytic cellsthat clear dying cells. Several macrophage receptors have beenimplicated in recognizing PS on apoptotic cells, including variousscavenger receptors, CD36, CD14, and PS receptor (PSR). Thus, PS has ademonstrated ability to mediate cell-cell interactions and to functionas a ligand for a variety of PS-binding receptors.

Enveloped viruses expose PS on their host-captured lipid bilayermembranes constantly. Enveloped viruses utilize this PS-exposure toevade attacks by the human immune system and to enter phagocytic cellslike monocytes/macrophages making its appearance in the viral membranehighly suspect as a factor in virus-target cell fusion.

Viruses that infect animals and humans infect cells by placing theirgenetic material within the cytoplasm and/or nucleoplasm of the infectedcell. “Response elements” within the genome, which may comprise codingregions or non-coding regions, respond to molecular signaling of thehost cell and/or other elements of the virus' own molecular network.Viruses often have “glucocorticoid response elements” (GRE), namelyresponse elements that are under the influence of glucocorticoidsignaling mediated by the binding of cortisol (or other glucocorticoids)to the glucocorticoid receptor (GCR).

This binding, which is activating, leads to signaling cascadesmodulating endogenous GRE of the host cell as well as viral GRE.Modulation of the viral GRE may directly or indirectly promote viralphysiology that will promote fundamental viral functions (including, butnot limited to genetic replication, production of virus-associatedproteins, assembly of genetic material and viral proteins into completeviruses, increasing genetic diversity, promotion of viral active orpassive virus release from the cell, and viral infectivity.

ORG 34517, PT150, PT155, PT156, PT157, PT158, and TCY1 are members of aclass of therapeutic agents designed to block the glucorticoid receptor(GR), acting as an antagonist for endogenous cortisol. Its primarydevelopmental pathway has been as a treatment for neuropsychiatricdiseases that are characterized by dysregulated signaling in thehypothalamic-pituitary-adrenal axis, often with higher than normalcirculating levels of endogenous cortisol. Of particular note are thephase 2 clinical trials that have been completed for the treatment ofpsychotic depression. Other possible uses in this disease category whichare under investigation include: post-traumatic stress disorder, weightgain in patients requiring long term anti-psychotic medication, hospitaldelirium of the elderly, etc.

The endogenous glucocorticoids are steroids predominantly produced inthe adrenal cortex. Glucocorticoids are important steroids forintermediary metabolism, immune, musculoskeletal, connective tissue andbrain function. The main glucocorticoid in the body is cortisol. Theproduction and secretion of cortisol is governed by a complex and highlyefficient system that includes the hypothalamus, pituitary and theadrenal glands i.e., hypothalamic-pituitary-adrenal axis (HPA). Cortisolsecretion has a circadian release rhythm with peak values in earlymorning and trough values at midnight.

The production and secretion of the most important glucocorticoid,cortisol, is governed by a complex and highly efficient system thatincludes the hypothalamus, pituitary and the adrenal glands i.e.,hypothalamic-pituitary-adrenal axis. Cortisol secretion is regulated bythe suprachiasmatic nucleus of the hypothalamus into a circadian releaserhythm. The timing is synchronized with the solar day by dark-lightshifts, which normally reflect the habitual sleep-wake pattern.Therefore in healthy persons, the cortisol secretion has a 24-hourcircadian pattern with peak serum levels in the early morning, 3-6 hoursafter onset of sleep, and nadir levels around midnight. Physical andpsychological stressors also activate cortisol secretion. Changedpatterns of serum cortisol levels have been observed in connection withabnormal adrenocorticotropic hormone (ACTH), levels, clinicaldepression, psychological stress, and physiological stressors such ashypoglycemia, illness, fever, trauma, surgery, fear, pain, physicalexertion, or temperature extremes. Cortisol levels and responsivenessmay also differ from normal for elderly individuals and in individualswith autism or Asperger's syndrome.

Glucocorticoids (GCs) such as, in humans, cortisol, perform severalimportant functions. These include participating in the regulation ofcarbohydrate, protein and fat metabolism by signaling the liver to makeglucose and glycogen, the adipose tissues to release lipids and fattyacids into the bloodstream, and the skeletal muscles to release proteinsor amino acids into the bloodstream. GCs also decrease bone formation.

GCs also regulate the body's inflammatory response as well. GCs are partof the feedback mechanism in the immune system that inhibits immuneactivity (i.e., inflammation). GCs cause their effects by binding to theGCR. The activated GCR complex in turn up-regulates the expression ofanti-inflammatory proteins in the nucleus (a process known astransactivation) and represses the expression of pro-inflammatoryproteins in the cytosol by preventing the translocation of othertranscription factors from the cytosol into the nucleus(transrepression) (Rhen T and Cidlowski J A. NEJM 2005; 353: 1711-23).

GCR antagonist or active agent therapy is helpful in patients withabnormally high levels of cortisol (but maintained circadian rhythm),over responsiveness to normal levels, or high night time cortisol levelsas a feature of disrupted circadian rhythm. Such altered cortisolphysiology may relate to acute or chronic stress (e.g. related tophysical or psychological trauma) or as an age related change in elderlyindividuals. Successful therapeutic use of such agents is thus oftendependent on determining circadian cortisol levels (either peak levelsduring the day, e.g., at noon, or measurements taken every 4 hours or 6hours over a 24 hour period). This combined system of salivary cortisolquantification as an enabling device for its paired GCR antagonist willidentify individuals for whom GCR antagonist or active agent therapy hasa benefit.

The glucocorticoid receptor (GR) is expressed at high levels in somenormal tissues, but not in others. Likewise, malignant tumors of diversetypes and sites have variable GR expression. When present in normal ortumor (benign or malignant) tissues, this GR expression may be variouslylocated in some or all of their cellular sub-compartments: 1. stemcells; 2. progenitor (so called “transit amplifying”) cell descendentsof activated stem cells; and 3. differentiated progeny of activated stemor progenitor cells.

The present invention therefore relates to the use of GR antagonists oractive agents (e.g., ORG34517, PT150—a relatively specific GRantagonist, RU486—a non-specific GR antagonist, and others), optionallyin combination with at least one other agent, for treating or preventingtreatment resistant prostate cancer, treating or preventing neoplasia,and/or treating or preventing infection related to acute or chronicinjury or disease.

ORG 34517, PT150, PT155, PT156, PT157, PT158, TCY1 are members of aclass of therapeutic agents designed to block the glucocorticoidreceptor (GR), acting as an antagonist for endogenous cortisol. Itsprimary developmental pathway has been as a treatment forneuropsychiatric diseases that are characterized by dysregulatedsignaling in the hypothalamic-pituitary-adrenal axis, often with higherthan normal circulating levels of endogenous cortisol. Of particularnote are the phase 2 clinical trials that have been completed for thetreatment of psychotic depression. Other possible uses in this diseasecategory which are under investigation include: post-traumatic stressdisorder, weight gain in patients requiring long term anti-psychoticmedication, hospital delirium of the elderly, etc. In addition, thediverse data indicate a possible role for GR-blockade as a means ofpromoting chemo-sensitization of target tumors. Pre-clinical trialsdemonstrate significant outcomes—breast cancer growth slowed andreversed. These are pre-clinical trials in which the company hassuccessfully demonstrated the efficacy of a chemotherapy sensitizer for“triple negative” breast cancer, ovarian cancer and prostate cancer.

The “triple negative” breast cancer is the most difficult to treat typeof breast cancer, and is indicated by the patient testing negative forestrogen-receptor, progesterone-receptor and her-2/neu. The triplenegative breast cancer is resistant to chemotherapy. Primary drugresistance and early onset of resistance are seen in other tumor types,as well for example in liver and ovarian cancers, where there is asignificant unmet medical need for effective therapy. Chemotherapy isstill a key approach to cancer treatment. Chemosensitizers wouldcontribute to improve the efficacy of current therapeutic drugs andpotentially improve their side effect profile. The world cancer marketwas estimated at $23 billion in 2004 and is expected to grow to at least$61 billion by 2013 with a CAGR of 14.7%.

The present invention provides a low cost rapid response diagnosticsystem to determine salivary cortisol levels in patients selected aspotential candidates for GCR (glucocorticoid receptor) antagonisttherapy utilizing a GCR antagonist or active agent such as ORG 34517,PT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof. The inventors have developeda saliva based diagnostic device for cortisol detection to accompany thedevelopment of ORG 34517, PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof as atherapeutic agent for multiple indications. Clinical testing of cortisollevels in patients is a high cost, laborious test that can be salivaryor serum, with samples taken from a patient and sent to a lab to awaitresults. The cost and time factor for such tests has, to date, beenprohibitive, preventing the rapid quantitative determination necessaryto assign treatment with a glucocorticoid receptor (GCR) antagonist dueto the inability to make the determinations of cortisol levels at pointof need or to monitor changes in cortisol as a measure of treatmentresponse. By allowing the physician to determine the elevated cortisollevel of a patient and in turn provide a therapeutic for such elevationat point of measurement, the physician can qualify the best candidatessuited for this type of therapeutic. The system also enables continualmonitoring of the patient during treatment for assessment ofresponsiveness to treatment.

The present invention provides a system in which an apparatus uses ahigh void volume carrier to absorb sufficient amounts of saliva to thenbe placed into a reaction vessel with a reagent. The reagent is mixedwith the sample and then is combined with, for example, a fluorescentligand or pigment-labeled ligand and placed into a device to determinesalivary cortisol levels of the patient in less than 5 minutes, ineither a portable, miniaturized fluorescence polarization reader (in theformer case) or into a lateral flow device (in the latter) for measuringamounts of substrate in a small amount of fluid by direct or indirectmethods.

The reader apparatus, for example, provides temperature control andon-board mixing as an aid in viscosity control of the reaction to ensurebetter accuracy and precision.

The invention and method for non-invasive sampling and detecting thepresence of a biological substance of interest in a test sample of, forexample, saliva, or a bodily fluid, combining said test sample with abuffering system (Reagent 1) containing viscosity controllers andstabilizers in a reaction vessel, mix solution well, combining said testsample and buffering system mixture with a fluorescence-labeled ligand(Reagent 2) to said biological substance (assay solution) in a reactionvessel, mix solution well, and detecting a change of the assay solutionin the fluorescence polarization reader, or a pigment labeled ligand.

The ongoing development of the present invention has yielded newfindings; the thiosemicarbazone of ORG34517 could not be dimerized bytreatment with sodium hydroxide NaOH. However, in-depth considerationsindicated that this is in fact better for the goal to eliminate humanhepatitis B and immunodeficiency proviruses, since the crucial point isthe binding mode on human glucocorticoid receptor (hGR). It could beshown that the anticipated dimer would not bind to hGR. Thethiosemicarbazone of ORG34517 could bind to hGR and force nucleartranslocation of the ligand-receptor complex. This is important, sincenuclear translocation is the prerequisite for our mode of action, andthe ORG34517-hGR complex itself does not translocate into the nucleus.In addition, the thiosemicarbazone of ORG34517 will be activated toreactive sulfenic acid and carbodiimide metabolites by humanflavin-containing monooxygenases (hFMO1, hFMO2.1, hFMO3). The activationis achieved not by an activated bond in a putative dimer, but bymetabolic activation with human enzymes. In addition, oxidative stressis enhanced in human hepatitis B and human immunodeficiencyvirus-infected cells, this might lead to enhanced activation invirus-infected cells. The material PT155 is the complex of choice to beused in antiviral studies in vitro. The present invention relates to theuse of glucocorticoid receptor (GCR) antagonists or active agents (e.g.ORG 34517, PT150, PT155, PT156, PT157, PT158, TCY1, combinationsthereof, and pharmaceutically acceptable salts thereof) enabled by adevice for rapidly, sensitively, specifically quantifying salivarycortisol levels as a surrogate for serum cortisol levels in a low costmanner. One purpose of this combination of inventions is to determinepatients who have non-normal cortisol produced by the adrenal cortex ordisordered circadian rhythms as a method for selecting subjects for GCRantagonist or active agent therapy for whom it is likely to havebeneficial and/or therapeutic effects, i.e., those with abnormal highlevels (but maintained circadian rhythm), over responsiveness to normallevels, high night-time cortisol levels as a feature of disruptedcircadian rhythm. The rapid, sensitive, and inexpensive test can also beused to monitor changes in cortisol levels in response to treatment, inpatients who have nonnormal cortisol produced by the adrenal cortex ordisordered circadian rhythms as a method for selecting subjects for GCRantagonist or active agent therapy for whom it is likely to havebeneficial and/or therapeutic effects, but also in patients havingnormal baseline cortisol at the start of treatment, but for whomchanging cortisol levels during treatment will indicate responsivenessto the GCR antagonist.

The endogenous glucocorticoids are steroids predominantly produced inthe adrenal cortex. Glucocorticoids are important steroids forintermediary metabolism, immune, musculoskeletal, connective tissue andbrain function. The main glucocorticoid in the body is cortisol. Theproduction and secretion of cortisol is governed by a complex and highlyefficient system that includes the hypothalamus, pituitary and theadrenal glands i.e., hypothalamic-pituitary-adrenal axis (HPA). Cortisolsecretion has a circadian release rhythm with peak values in earlymorning and trough values at midnight.

The production and secretion of the most important glucocorticoid,cortisol, is governed by a complex and highly efficient system thatincludes the hypothalamus, pituitary and the adrenal glands i.e.,hypothalamic-pituitary-adrenal axis. Cortisol secretion is regulated bythe suprachiasmatic nucleus of the hypothalamus into a circadian releaserhythm. The timing is synchronized with the solar day by dark-lightshifts, which normally reflect the habitual sleep-wake pattern.Therefore in healthy persons, the cortisol secretion has a 24-hourcircadian pattern with peak serum levels in the early morning, 3-6 hoursafter onset of sleep, and nadir levels around midnight. Physical andpsychological stressors also activate cortisol secretion. Changedpatterns of serum cortisol levels have been observed in connection withabnormal adrenocorticotropic hormone (ACTH), levels, clinicaldepression, psychological stress, and physiological stressors such ashypoglycemia, illness, fever, trauma, surgery, fear, pain, physicalexertion, or temperature extremes. Cortisol levels and responsivenessmay also differ from normal for elderly individuals and in individualswith autism or Asperger's syndrome.

Glucocorticoids (GCs) such as, in humans, cortisol, perform severalimportant functions. These include participating in the regulation ofcarbohydrate, protein and fat metabolism by signaling the liver to makeglucose and glycogen, the adipose tissues to release lipids and fattyacids into the bloodstream, and the skeletal muscles to release proteinsor amino acids into the bloodstream. GCs also decrease bone formation.

GCs also regulate the body's inflammatory response as well. GCs are partof the feedback mechanism in the immune system that inhibits immuneactivity (i.e., inflammation). GCs cause their effects by binding to theGCR. The activated GCR complex in turn up-regulates the expression ofanti-inflammatory proteins in the nucleus (a process known astransactivation) and represses the expression of pro-inflammatoryproteins in the cytosol by preventing the translocation of othertranscription factors from the cytosol into the nucleus(transrepression) (Rhen T and Cidlowski J A. NEJM 2005; 353: 1711-23).

GCR antagonist or active agent therapy is helpful in patients withabnormally high levels of cortisol (but maintained circadian rhythm),over responsiveness to normal levels, or high night time cortisol levelsas a feature of disrupted circadian rhythm. Successful therapeutic useof such agents is thus dependent on determining circadian cortisollevels (either peak levels during the day, e.g., at noon, ormeasurements taken every 4 hours or 6 hours over a 24 hour period). Thiscombined system of salivary cortisol quantification as an enablingdevice for its paired GCR antagonist will identify individuals for whomGCR antagonist or active agent therapy has a benefit.

The glucocorticoid receptor (GR) is expressed at high levels in somenormal tissues, but not in others. Likewise, malignant tumors of diversetypes and sites have variable GR expression. When present in normal ortumor (benign or malignant) tissues, this GR expression may be variouslylocated in some or all of their cellular subcompartments: 1. stem cells;2. progenitor (so called “transit amplifying”) cell descendents ofactivated stem cells; and 3. differentiated progeny of activated stem orprogenitor cells.

As an example, in the gastrointestinal tract, GR are highly expressed inesophageal squamous epithelia, hepatocytes, and pancreatic islet cells,but are not highly expressed in other gastrointestinal epithelia(stomach, small and large intestines, pancreatic and biliary ducts). Incorresponding malignancies arising in these epithelia, hepatocellularcarcinoma (HCC) and squamous cell carcinomas (SCC) of the esophagus haveconsistently highGR expression. Gastric and colorectal adenocarcinomashave little to no GR expression.

Dexamethasone (DEX), a binding activator of GR, has been found to conferchemoresi stance in oesophageal SCC and HCC cells, suggesting that GRexpression may be biologically important in some GR-expressingcarcinomas. This not only suggests why DEX or other glucocorticoids arenot useful in treatment of these malignancies, but it implies thatendogenous, circulating cortisol itself may actually promote chemoresistance, even in the absence of iatrogenic glucocorticoid administration.Therefore, these findings suggest that blockade of GR within suchmalignant tumors, by preventing activation by endogenous, circulatingcortisol, can play a role in maintaining or promoting chemosensitivityand/or treating neoplasia.

The present invention therefore relates to the use of GR antagonists oractive agents (e.g., ORG 34517, PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof) forthe treatment of, for example, esophageal SCC and HCC or other tumorswith high GR expression as a means of inhibiting promotion of chemoresistance by endogenous cortisol. These effects may be present in all tumorcells or, when tumors have stem or progenitor cell compartments, these,specifically, as well. Thus, the present invention relates to theinhibition of chemoprevention in the bulk of cells making up a giventumor and/or in the rare stem/progenitor cells within the tumor that areoften responsible for tumor resistance to therapy and reoccurrence,i.e., as a novel, targeted “cancer stem cell” treatment.

To avoid possible negative side effects of systemic blockade of GR, thepresent invention further relates to localized tumor treatment with GRantagonists through direct vascular infusion of tumor feeding vessels orby direct, intratumoral injection.

The present invention relates to the use of GR antagonists for thetreatment of, for example, breast and other cancers. The invention isbased on the observation that GR inhibition will increase tumor cellsusceptibility. GR antagonists will block anti-apoptotic GR signaling inGR-overexpressing breast cancer cells and subsequently render breastcancer cells more susceptible to conventional and novel cytotoxictherapies (via blocking GR's pro-cell survival signaling pathway).

All references cited herein are incorporated herein by reference intheir entireties.

BRIEF SUMMARY OF THE INVENTION

The invention provides a compound selected from the group consisting of:

or pharmaceutically acceptable salts thereof;

or pharmaceutically acceptable salts thereof;

or pharmaceutically acceptable salts thereof;

or pharmaceutically acceptable salts thereof;

or pharmaceutically acceptable salts thereof; and, combinations thereof.The invention provides a pharmaceutical composition comprising atherapeutically effective amount of at least one active agent, whereinthe active agent is selected from the group consisting of:

or pharmaceutically acceptable salts thereof;

or pharmaceutically acceptable salts thereof;

or pharmaceutically acceptable salts thereof;

or pharmaceutically acceptable salts thereof;

or pharmaceutically acceptable salts thereof; combinations thereof;optionally, at least one additional pharmaceutically active agent; andat least one pharmaceutically acceptable excipient.

The invention provides a pharmaceutical composition in a dosage formselected from the group consisting of a minicapsule, a capsule, atablet, an implant, a troche, a lozenge, a minitablet, a temporary orpermanent suspension, an injectable, an ovule, a suppository, a wafer, achewable tablet, a quick or fast dissolving tablet, an effervescenttablet, a buccal or sublingual solid, a granule, a film, a sprinkle, apellet, a topical formulation, a patch, a bead, a pill, a powder, atriturate, a smart pill, a smart capsule, a platelet, a strip, and asachet.

The invention provides a pharmaceutical composition in a dosage form fortopical application, and at least one pharmaceutically acceptableexcipient. The invention provides a pharmaceutical composition in adosage form for topical application wherein said formulation is in aform selected from the group consisting of: cream, lotion, gel, oil,ointment, suppository, spray, foam, liniment, aerosol, buccal andsublingual tablet or a transdermal device or patch for absorptionthrough the skin or mucous membranes.

The invention provides a kit for treating or preventing a condition in apatient, the kit comprising: (a) the pharmaceutical composition in atherapeutically effective amount; and (b) at least one blister package;a lidded blister; a blister card or packet; a clamshell; an intravenous(IV) package, IV packette or IV container; a tray or a shrink wrapcomprising the pharmaceutical composition of (a) and instructions forusing the pharmaceutical composition.

The invention provides a method of treating and/or preventing a viralcondition in a patient comprising: selecting a patient in need oftreating and/or preventing a viral condition; administering to thepatient at least one active agent selected from the group consisting ofPT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof, wherein the viral conditionis to prevent or eliminate acute viral infection, to diminish intensityof viral infection, to diminish length of viral infection, to speed timeto resolution and healing of viral infection, to speed time tosuppression of viral infection, to increase likelihood of viraleradication, and/or to diminish infectivity of viral infection, withHepatitis C virus, Bovine Viral Diarrhea virus, Ebola-like viruses,Hepatitis B virus, Mouse mammary tumor virus, Human ImmunodeficiencyVirus-1 (HIV-1), Varicella-Zoster virus (chicken pox; VZV),Cytomegalovirus (CMV), Human Herpes Virus-6 (HHV-6), Human HerpesVirus-7 (HHV-7), Kaposi's Sarcoma-Associated Herpes virus (or HumanHerpes Virus-8; HHV-8), Variola (Small Pox) virus, Vaccinia virus,Cowpox virus, Monkeypox virus.

The invention provides a method of treating and/or preventing a viralcondition in a patient comprising: selecting the patient in need oftreating and/or preventing a viral condition; administering at least oneactive agent selected from the group consisting of PT150, PT155, PT156,PT157, PT158, TCY1, combinations thereof, and pharmaceuticallyacceptable salts thereof, wherein the viral condition is to preventacute viral infection from becoming chronic active or latent infectionwith Hepatitis C virus, Bovine Viral Diarrhea virus, Ebola-like viruses,Hepatitis B virus, Mouse mammary tumor virus, Human ImmunodeficiencyVirus-1 (HIV-1), Varicella-Zoster virus (chicken pox; VZV),Cytomegalovirus (CMV), Human Herpes Virus-6 (HHV-6), Human HerpesVirus-7 (HHV-7), Kaposi's Sarcoma-Associated Herpes virus (or HumanHerpes Virus-8; HHV-8), Variola (Small Pox) virus, Vaccinia virus,Cowpox virus, Monkeypox virus.

The invention provides a method of treating and/or preventing a viralcondition in a patient comprising: selecting a patient in need oftreating and/or preventing a viral condition; administering at least oneactive agent selected from the group consisting of PT150, PT155, PT156,PT157, PT158, TCY1, combinations thereof, and pharmaceuticallyacceptable salts thereof, wherein the viral condition is to preventchronic latent viral infection from becoming active (reactivation), todiminish intensity of viral reactivation, to diminish length of viralreactivation, to speed time to resolution and healing of viralreactivation, to speed time to suppression of viral reactivation, toincrease likelihood of viral eradication, and/or to diminish infectivityof viral reactivation with: Hepatitis C virus, Bovine Viral Diarrheavirus, Ebola-like viruses, Hepatitis B virus, Mouse mammary tumor virus,Human Immunodeficiency Virus-1 (HIV-1), Varicella-Zoster virus (chickenpox; VZV), Cytomegalovirus (CMV), Human Herpes Virus-6 (HHV-6), HumanHerpes Virus-7 (HHV-7), Kaposi's Sarcoma-Associated Herpes virus (orHuman Herpes Virus-8; HHV-8), Variola (Small Pox) virus, Vaccinia virus,Cowpox virus, Monkeypox virus.

The invention provides a method of treating and/or preventing a viralcondition in a patient comprising: selecting a patient in need oftreating and/or preventing a viral condition; administering at least oneactive agent selected from the group consisting of PT150, PT155, PT156,PT157, PT158, TCY1, combinations thereof, and pharmaceuticallyacceptable salts thereof, wherein the viral condition is to inactivatelatent pro-viral genome eliminating (“curing”) chronic viral infectionswith Hepatitis C virus, Bovine Viral Diarrhea virus, Ebola-like viruses,Hepatitis B virus, Mouse mammary tumor virus, Human ImmunodeficiencyVirus-1 (HIV-1), Varicella-Zoster virus (chicken pox; VZV),Cytomegalovirus (CMV), Human Herpes Virus-6 (HHV-6), Human HerpesVirus-7 (HHV-7), Kaposi's Sarcoma-Associated Herpes virus (or HumanHerpes Virus-8; HHV-8), Variola (Small Pox) virus, Vaccinia virus,Cowpox virus, Monkeypox virus.

The invention provides a method of treating and/or preventing a viralcondition in a patient comprising: selecting a patient in need oftreating and/or preventing a viral condition; administering an activeagent selected from the group consisting of PT150, PT155, PT156, PT157,PT158, TCY1, combinations thereof, and pharmaceutically acceptable saltsthereof, wherein the viral condition is to prevent or eliminate acuteviral infection, to diminish intensity of viral infection, to diminishlength of viral infection, to speed time to resolution and healing ofviral infection, to speed time to suppression of viral infection, toincrease likelihood of viral eradication, and/or to diminish infectivityof viral infection. The invention provides a method wherein the viralcondition is selected from the group consisting of Ebola and Marburgvirus (Filoviridae); Ross River virus, chikungunya virus, Sindbis virus,eastern equine encephalitis virus (Togaviridae, Alphavirus), vesicularstomatitis virus (Rhabdoviridae, Vesiculovirus), Amapari virus, Pichindévirus, Tacaribe virus, Junin virus, Machupo virus (Arenaviridae,Mammarenavirus), West Nile virus, dengue virus, yellow fever virus(Flaviviridae, Flavivirus); human immunodeficiency virus type 1(Retroviridae, Lentivirus); Moloney murine leukemia virus (Retroviridae,Gammaretrovirus); influenza A virus (Orthomyxoviridae); respiratorysyncytial virus (Paramyxoviridae, Pneumovirinae, Pneumovirus); vacciniavirus (Poxviridae, Chordopoxvirinae, Orthopoxvirus); herpes simplexvirus type 1, herpes simplex virus type 2 (Herpesviridae,Alphaherpesvirinae, Simplexvirus); human cytomegalovirus (Herpesviridae,Betaherpesvirinae, Cytomegalovirus); Autographa californicanucleopolyhedrovirus (Baculoviridae, Alphabaculoviridae) (an insectvirus); Ebola and Marburg virus (Filoviridae); Semliki Forest virus,Ross River virus, chikungunya virus, O'nyong-nyong virus, Sindbis virus,eastern/western/Venezuelan equine encephalitis virus (Togaviridae,Alphavirus); rubella (German measles) virus (Togaviridae, Rubivirus);rabies virus, Lagos bat virus, Mokola virus (Rhabdoviridae, Lyssavirus);Amapari virus, Pichindé virus, Tacaribe virus, Junin virus, Machupovirus, Guanarito virus, Sabia virus, Lassa virus (Arenaviridae,Mammarenavirus); West Nile virus, dengue virus, yellow fever virus, Zikavirus, Japanese encephalitis virus, St. Louis encephalitis virus,tick-borne encephalitis virus, Omsk hemorrhagic fever virus, KyasanurForest virus (Flaviviridae, Flavivirus); human hepatitis C virus(Flaviviridae, Hepacivirus); human immunodeficiency virus type 1(Retroviridae, Lentivirus); influenza A/B virus (Orthomyxoviridae, thecommon ‘flu’ virus); respiratory syncytial virus (Paramyxoviridae,Pneumovirinae, Pneumovirus); Hendra virus, Nipah virus (Paramyxoviridae,Paramyxovirinae, Henipavirus); measles virus (Paramyxoviridae,Paramyxovirinae, Morbillivirus); variola major (smallpox) virus(Poxviridae, Chordopoxvirinae, Orthopoxvirus); human hepatitis B virus(Hepadnaviridae, Orthohepadnavirus); hepatitis delta virus (hepatitis Dvirus) (unassigned Family, Deltavirus); herpes simplex virus type 1,herpes simplex virus type 2 (Herpesviridae, Alphaherpesvirinae,Simplexvirus); human cytomegalovirus (Herpesviridae, Betaherpesvirinae,Cytomegalovirus).

The invention provides a method of treating and/or preventing a viralcondition in a patient comprising: selecting a patient in need oftreating and/or preventing a viral condition; administering to thepatient at least one active agent selected from the group consisting ofPT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof, wherein the viral conditionis to prevent acute viral infection from becoming chronic active orlatent infection. The invention provides a method wherein the viralcondition is selected from the group consisting of Ebola and Marburgvirus (Filoviridae); Ross River virus, chikungunya virus, Sindbis virus,eastern equine encephalitis virus (Togaviridae, Alphavirus), vesicularstomatitis virus (Rhabdoviridae, Vesiculovirus), Amapari virus, Pichindévirus, Tacaribe virus, Junin virus, Machupo virus (Arenaviridae,Mammarenavirus), West Nile virus, dengue virus, yellow fever virus(Flaviviridae, Flavivirus); human immunodeficiency virus type 1(Retroviridae, Lentivirus); Moloney murine leukemia virus (Retroviridae,Gammaretrovirus); influenza A virus (Orthomyxoviridae); respiratorysyncytial virus (Paramyxoviridae, Pneumovirinae, Pneumovirus); vacciniavirus (Poxviridae, Chordopoxvirinae, Orthopoxvirus); herpes simplexvirus type 1, herpes simplex virus type 2 (Herpesviridae,Alphaherpesvirinae, Simplexvirus); human cytomegalovirus (Herpesviridae,Betaherpesvirinae, Cytomegalovirus); Autographa californicanucleopolyhedrovirus (Baculoviridae, Alphabaculoviridae) (an insectvirus); Ebola and Marburg virus (Filoviridae); Semliki Forest virus,Ross River virus, chikungunya virus, O'nyong-nyong virus, Sindbis virus,eastern/western/Venezuelan equine encephalitis virus (Togaviridae,Alphavirus); rubella (German measles) virus (Togaviridae, Rubivirus);rabies virus, Lagos bat virus, Mokola virus (Rhabdoviridae, Lyssavirus);Amapari virus, Pichindé virus, Tacaribe virus, Junin virus, Machupovirus, Guanarito virus, Sabia virus, Lassa virus (Arenaviridae,Mammarenavirus); West Nile virus, dengue virus, yellow fever virus, Zikavirus, Japanese encephalitis virus, St. Louis encephalitis virus,tick-borne encephalitis virus, Omsk hemorrhagic fever virus, KyasanurForest virus (Flaviviridae, Flavivirus); human hepatitis C virus(Flaviviridae, Hepacivirus); human immunodeficiency virus type 1(Retroviridae, Lentivirus); influenza A/B virus (Orthomyxoviridae, thecommon ‘flu’ virus); respiratory syncytial virus (Paramyxoviridae,Pneumovirinae, Pneumovirus); Hendra virus, Nipah virus (Paramyxoviridae,Paramyxovirinae, Henipavirus); measles virus (Paramyxoviridae,Paramyxovirinae, Morbillivirus); variola major (smallpox) virus(Poxviridae, Chordopoxvirinae, Orthopoxvirus); human hepatitis B virus(Hepadnaviridae, Orthohepadnavirus); hepatitis delta virus (hepatitis Dvirus) (unassigned Family, Deltavirus); herpes simplex virus type 1,herpes simplex virus type 2 (Herpesviridae, Alphaherpesvirinae,Simplexvirus); human cytomegalovirus (Herpesviridae, Betaherpesvirinae,Cytomegalovirus).

The invention provides a pharmaceutical composition comprising atherapeutically effective amount of any one or more of a compoundselected from the group consisting of PT150, PT155, PT156, PT157, PT158,TCY1, combinations thereof, and pharmaceutically acceptable saltsthereof; at least one additional active agent selected from the groupconsisting of molecules with potential to bind viral PS, annexin-5,anti-PS monoclonal or polyclonal antibodies, bavituximab, and/or bind toviral glucocorticoid response elements (GREs), retinazone, RU486, ortheir derivatives; and at least one pharmaceutically acceptable carrier;optionally, at least one blister package; a lidded blister; a blistercard or packet; a clamshell; an intravenous (IV) package, IV packette orIV container; a tray or a shrink wrap comprising the pharmaceuticalcomposition and instructions for use of the pharmaceutical composition.

The invention provides a pharmaceutical composition comprising: atherapeutically effective amount of one or more of PT150, PT155, PT156,PT157, PT158, TCY1, combinations thereof, and pharmaceuticallyacceptable salts thereof; at least one additional active agent selectedfrom the group consisting of molecules with potential to bind viral PS,annexin-5, anti-PS monoclonal or polyclonal antibodies, bavituximab,and/or bind to viral glucocorticoid response elements (GREs), retinazoneand RU486 or derivatives, cell entry inhibitors, uncoating inhibitors,reverse transcriptase inhibitors, integrase inhibitors, transcriptioninhibitors, antisense translation inhibitors, ribozyme translationinhibitors, prein processing and targeting inhibitors, proteaseinhibitors, assembly inhibitors, release phase inhibitos, immunosystemmodulators and vaccines, including, but not limited to Abacavir, Ziagen,Trizivir, Kivexa/Epzicom, Aciclovir, Acyclovir, Adefovir, Amantadine,Amprenavir, Ampligen, Arbidol, Atazanavir, Atripla, Balavir, Cidofovir,Combivir, Dolutegravir, Darunavir, Delavirdine, Didanosine, Docosanol,Edoxudine, Efavirenz, Emtricitabine, Enfuvirtide, Entecavir, Ecoliever,Famciclovir, Fomivirsen, Fosamprenavir, Foscarnet, Fosfonet,Ganciclovir, Ibacitabine, Imunovir, Idoxuridine, Imiquimod, Indinavir,Inosine, Integrase inhibitor, Interferon type III, Interferon type II,Interferon type I, Interferon, Lamivudine, Lopinavir, Loviride,Maraviroc, Moroxydine, Methisazone, Nelfinavir, Nevirapine, Nexavir,Nucleoside analogues, Novir, Oseltamivir (Tamiflu), Peginterferonalfa-2a, Penciclovir, Peramivir, Pleconaril, Podophyllotoxin, Proteaseinhibitor, Raltegravir, Reverse transcriptase inhibitor, Ribavirin,Rimantadine, Ritonavir, Pyramidine, Saquinavir, Sofosbuvir, Stavudine,Synergistic enhancer, Tea tree oil, Telaprevir, Tenofovir, Tenofovirdisoproxil, Tipranavir, Trifluridine, Trizivir, Tromantadine, Truvada,Valaciclovir, Valganciclovir, Vicriviroc, Vidarabine, Viramidine,Zalcitabine, Zanamivir, Zidovudine, and combinations thereof; at leastone pharmaceutically acceptable carrier; and optionally, and at leastone blister package; a lidded blister; a blister card or packet; aclamshell; an intravenous (IV) package, IV packette or IV container; atray or a shrink wrap comprising the pharmaceutical composition andinstructions for use of the pharmaceutical composition.

The invention provides a method of treating and/or preventing a viralcondition in a patient comprising: selecting a patient in need oftreating and/or preventing a viral condition; administering to thepatient at least one active agent selected from the group consisting ofPT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof; administering at least oneadditional active agent selected from the group consisting of othermolecules with potential to bind viral glucocorticoid response elements(GREs), retinazone, RU486, derivatives thereof, wherein the viralcondition is to prevent or eliminate acute viral infection, to diminishintensity of viral infection, to diminish length of viral infection, tospeed time to resolution and healing of viral infection, to speed timeto suppression of viral infection, to increase likelihood of viraleradication, and/or to diminish infectivity of viral infection, withHepatitis C virus, Bovine Viral Diarrhea virus, Ebola-like viruses,Hepatitis B virus, Mouse mammary tumor virus, Human ImmunodeficiencyVirus-1 (HIV-1), Varicella-Zoster virus (chicken pox; VZV),Cytomegalovirus (CMV), Human Herpes Virus-6 (HHV-6), Human HerpesVirus-7 (HHV-7), Kaposi's Sarcoma-Associated Herpes virus (or HumanHerpes Virus-8; HHV-8), Variola (Small Pox) virus, Vaccinia virus,Cowpox virus, Monkeypox virus.

The invention provides a method of treating and/or preventing a viralcondition in a patient comprising: selecting the patient in need oftreating and/or preventing a viral condition; administering at least oneactive agent selected from the group consisting of PT150, PT155, PT156,PT157, PT158, TCY1, combinations thereof, and pharmaceuticallyacceptable salts thereof; administering at least one additional activeagent selected from the group consisting of molecules with potential tobind viral glucocorticoid response elements (GREs), retinazone, RU486,and derivatives thereof, wherein the viral condition is to prevent acuteviral infection from becoming chronic active or latent infection withHepatitis C virus, Bovine Viral Diarrhea virus, Ebola-like viruses,Hepatitis B virus, Mouse mammary tumor virus, Human ImmunodeficiencyVirus-1 (HIV-1), Varicella-Zoster virus (chicken pox; VZV),Cytomegalovirus (CMV), Human Herpes Virus-6 (HHV-6), Human HerpesVirus-7 (HHV-7), Kaposi's Sarcoma-Associated Herpes virus (or HumanHerpes Virus-8; HHV-8), Variola (Small Pox) virus, Vaccinia virus,Cowpox virus, Monkeypox virus.

The invention provides a method of treating and/or preventing a viralcondition in a patient comprising: selecting a patient in need oftreating and/or preventing a viral condition; administering at least oneactive agent selected from the group consisting of PT150, PT155, PT156,PT157, PT158, TCY1, combinations thereof, and pharmaceuticallyacceptable salts thereof; administering at least one additional activeagent selected from the group consisting of molecules with potential tobind viral glucocorticoid response elements (GREs), retinazone, RU486,or their derivatives, wherein the viral condition is to prevent chroniclatent viral infection from becoming active (reactivation), to diminishintensity of viral reactivation, to diminish length of viralreactivation, to speed time to resolution and healing of viralreactivation, to speed time to suppression of viral reactivation, toincrease likelihood of viral eradication, and/or to diminish infectivityof viral reactivation with: Hepatitis C virus, Bovine Viral Diarrheavirus, Ebola-like viruses, Hepatitis B virus, Mouse mammary tumor virus,Human Immunodeficiency Virus-1 (HIV-1), Varicella-Zoster virus (chickenpox; VZV), Cytomegalovirus (CMV), Human Herpes Virus-6 (HHV-6), HumanHerpes Virus-7 (HHV-7), Kaposi's Sarcoma-Associated Herpes virus (orHuman Herpes Virus-8; HHV-8), Variola (Small Pox) virus, Vaccinia virus,Cowpox virus, Monkeypox virus.

The invention provides a method of treating and/or preventing a viralcondition in a patient comprising: selecting a patient in need oftreating and/or preventing a viral condition; administering at least oneactive agent selected from the group consisting of PT150, PT155, PT156,PT157, PT158, TCY1, combinations thereof, and pharmaceuticallyacceptable salts thereof; administering at least one additional activeagent selected from the group consisting of molecules with potential tobind viral glucocorticoid response elements (GREs), retinazone, RU486 ortheir derivatives wherein the viral condition is to inactivate latentpro-viral genome eliminating (“curing”) chronic viral infections withHepatitis C virus, Bovine Viral Diarrhea virus, Ebola-like viruses,Hepatitis B virus, Mouse mammary tumor virus, Human ImmunodeficiencyVirus-1 (HIV-1), Varicella-Zoster virus (chicken pox; VZV),Cytomegalovirus (CMV), Human Herpes Virus-6 (HHV-6), Human HerpesVirus-7 (HHV-7), Kaposi's Sarcoma-Associated Herpes virus (or HumanHerpes Virus-8; HHV-8), Variola (Small Pox) virus, Vaccinia virus,Cowpox virus, Monkeypox virus.

The invention provides a method of treating and/or preventing a viralcondition in a patient comprising: selecting a patient in need oftreating and/or preventing a viral condition; administering an activeagent selected from the group consisting of PT150, PT155, PT156, PT157,PT158, TCY1, combinations thereof, and pharmaceutically acceptable saltsthereof; administering at least one additional active agent selectedfrom the group consisting of molecules with potential to bind viral PS,annexin-5, anti-PS monoclonal or polyclonal antibodies, bavituximab,wherein the viral condition is to prevent or eliminate acute viralinfection, to diminish intensity of viral infection, to diminish lengthof viral infection, to speed time to resolution and healing of viralinfection, to speed time to suppression of viral infection, to increaselikelihood of viral eradication, and/or to diminish infectivity of viralinfection. The invention provides a method wherein the viral conditionis selected from the group consisting of Ebola and Marburg virus(Filoviridae); Ross River virus, chikungunya virus, Sindbis virus,eastern equine encephalitis virus (Togaviridae, Alphavirus), vesicularstomatitis virus (Rhabdoviridae, Vesiculovirus), Amapari virus, Pichindévirus, Tacaribe virus, Junin virus, Machupo virus (Arenaviridae,Mammarenavirus), West Nile virus, dengue virus, yellow fever virus(Flaviviridae, Flavivirus); human immunodeficiency virus type 1(Retroviridae, Lentivirus); Moloney murine leukemia virus (Retroviridae,Gammaretrovirus); influenza A virus (Orthomyxoviridae); respiratorysyncytial virus (Paramyxoviridae, Pneumovirinae, Pneumovirus); vacciniavirus (Poxviridae, Chordopoxvirinae, Orthopoxvirus); herpes simplexvirus type 1, herpes simplex virus type 2 (Herpesviridae,Alphaherpesvirinae, Simplexvirus); human cytomegalovirus (Herpesviridae,Betaherpesvirinae, Cytomegalovirus); Autographa californicanucleopolyhedrovirus (Baculoviridae, Alphabaculoviridae) (an insectvirus); Ebola and Marburg virus (Filoviridae); Semliki Forest virus,Ross River virus, chikungunya virus, O'nyong-nyong virus, Sindbis virus,eastern/western/Venezuelan equine encephalitis virus (Togaviridae,Alphavirus); rubella (German measles) virus (Togaviridae, Rubivirus);rabies virus, Lagos bat virus, Mokola virus (Rhabdoviridae, Lyssavirus);Amapari virus, Pichindé virus, Tacaribe virus, Junin virus, Machupovirus, Guanarito virus, Sabia virus, Lassa virus (Arenaviridae,Mammarenavirus); West Nile virus, dengue virus, yellow fever virus, Zikavirus, Japanese encephalitis virus, St. Louis encephalitis virus,tick-borne encephalitis virus, Omsk hemorrhagic fever virus, KyasanurForest virus (Flaviviridae, Flavivirus); human hepatitis C virus(Flaviviridae, Hepacivirus); human immunodeficiency virus type 1(Retroviridae, Lentivirus); influenza A/B virus (Orthomyxoviridae, thecommon ‘flu’ virus); respiratory syncytial virus (Paramyxoviridae,Pneumovirinae, Pneumovirus); Hendra virus, Nipah virus (Paramyxoviridae,Paramyxovirinae, Henipavirus); measles virus (Paramyxoviridae,Paramyxovirinae, Morbillivirus); variola major (smallpox) virus(Poxviridae, Chordopoxvirinae, Orthopoxvirus); human hepatitis B virus(Hepadnaviridae, Orthohepadnavirus); hepatitis delta virus (hepatitis Dvirus) (unassigned Family, Deltavirus); herpes simplex virus type 1,herpes simplex virus type 2 (Herpesviridae, Alphaherpesvirinae,Simplexvirus); human cytomegalovirus (Herpesviridae, Betaherpesvirinae,Cytomegalovirus)

The invention provide s a method of treating and/or preventing a viralcondition in a patient comprising: selecting a patient in need oftreating and/or preventing a viral condition; administering to thepatient at least one active agent selected from the group consisting ofPT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof; administering at least oneadditional active agent selected from the group consisting of moleculeswith potential to bind viral PS, annexin-5, anti-PS monoclonal orpolyclonal antibodies, bavituximab, wherein the viral condition is toprevent acute viral infection from becoming chronic active or latentinfection. The invention provides a method wherein the viral conditionis selected from the group consisting of Ebola and Marburg virus(Filoviridae); Ross River virus, chikungunya virus, Sindbis virus,eastern equine encephalitis virus (Togaviridae, Alphavirus), vesicularstomatitis virus (Rhabdoviridae, Vesiculovirus), Amapari virus, Pichindévirus, Tacaribe virus, Junin virus, Machupo virus (Arenaviridae,Mammarenavirus), West Nile virus, dengue virus, yellow fever virus(Flaviviridae, Flavivirus); human immunodeficiency virus type 1(Retroviridae, Lentivirus); Moloney murine leukemia virus (Retroviridae,Gammaretrovirus); influenza A virus (Orthomyxoviridae); respiratorysyncytial virus (Paramyxoviridae, Pneumovirinae, Pneumovirus); vacciniavirus (Poxviridae, Chordopoxvirinae, Orthopoxvirus); herpes simplexvirus type 1, herpes simplex virus type 2 (Herpesviridae,Alphaherpesvirinae, Simplexvirus); human cytomegalovirus (Herpesviridae,Betaherpesvirinae, Cytomegalovirus); Autographa californicanucleopolyhedrovirus (Baculoviridae, Alphabaculoviridae) (an insectvirus); Ebola and Marburg virus (Filoviridae); Semliki Forest virus,Ross River virus, chikungunya virus, O'nyong-nyong virus, Sindbis virus,eastern/western/Venezuelan equine encephalitis virus (Togaviridae,Alphavirus); rubella (German measles) virus (Togaviridae, Rubivirus);rabies virus, Lagos bat virus, Mokola virus (Rhabdoviridae, Lyssavirus);Amapari virus, Pichindé virus, Tacaribe virus, Junin virus, Machupovirus, Guanarito virus, Sabia virus, Lassa virus (Arenaviridae,Mammarenavirus); West Nile virus, dengue virus, yellow fever virus, Zikavirus, Japanese encephalitis virus, St. Louis encephalitis virus,tick-borne encephalitis virus, Omsk hemorrhagic fever virus, KyasanurForest virus (Flaviviridae, Flavivirus); human hepatitis C virus(Flaviviridae, Hepacivirus); human immunodeficiency virus type 1(Retroviridae, Lentivirus); influenza A/B virus (Orthomyxoviridae, thecommon ‘flu’ virus); respiratory syncytial virus (Paramyxoviridae,Pneumovirinae, Pneumovirus); Hendra virus, Nipah virus (Paramyxoviridae,Paramyxovirinae, Henipavirus); measles virus (Paramyxoviridae,Paramyxovirinae, Morbillivirus); variola major (smallpox) virus(Poxviridae, Chordopoxvirinae, Orthopoxvirus); human hepatitis B virus(Hepadnaviridae, Orthohepadnavirus); hepatitis delta virus (hepatitis Dvirus) (unassigned Family, Deltavirus); herpes simplex virus type 1,herpes simplex virus type 2 (Herpesviridae, Alphaherpesvirinae,Simplexvirus); human cytomegalovirus (Herpesviridae, Betaherpesvirinae,Cytomegalovirus)

The invention provides a pharmaceutical composition comprising: at leastone active agent selected from the group consisting of PT150, PT155,PT156, PT157, PT158, TCY1, combinations thereof, and pharmaceuticallyacceptable salts thereof; optionally, at least one auxiliary agentselected from the group consisting of opiates. The invention provides apharmaceutical composition wherein the composition is in a dosage formselected from the group consisting of a capsule, a tablet, a smart pilldelivery device, and a smart capsule delivery device.

The invention provides a pharmaceutical composition comprising atherapeutically effective amount of one or more of PT150, PT155, PT156,PT157, PT158, TCY1, combinations thereof, and pharmaceuticallyacceptable salts thereof; at least one additional therapeutically activeagent selected from the group consisting of molecules with potential tobind viral PS, annexin-5, anti-PS monoclonal or polyclonal antibodies,bavituximab, and/or bind to viral glucocorticoid response elements(GREs), (including, but not limited to retinazone and RU486, derivativesthereof; and optionally at least one further active agent selected fromthe group consisting of anti-viral medications, cell entry inhibitors,uncoating inhibitors, reverse transcriptase inhibitors, integraseinhibitors, transcription inhibitors, translation (anti sense)inhibitors, translation (ribozyme) inhibitors, prein processing andtargeting inhibitors, protease inhibitors, assembly inhibitors, releasephase inhibitos, immunosystem modulators and vaccines, including, butnot limited to Abacavir, Ziagen, Trizivir, Kivexa/Epzicom, Aciclovir,Acyclovir, Adefovir, Amantadine, Amprenavir, Ampligen, Arbidol,Atazanavir, Atripla, Balavir, Cidofovir, Combivir, Dolutegravir,Darunavir, Delavirdine, Didanosine, Docosanol, Edoxudine, Efavirenz,Emtricitabine, Enfuvirtide, Entecavir, Ecoliever, Famciclovir,Fomivirsen, Fosamprenavir, Foscarnet, Fosfonet, Ganciclovir,Ibacitabine, Imunovir, Idoxuridine, Imiquimod, Indinavir, Inosine,Integrase inhibitor, Interferon type III, Interferon type II, Interferontype I, Interferon, Lamivudine, Lopinavir, Loviride, Maraviroc,Moroxydine, Methisazone, Nelfinavir, Nevirapine, Nexavir, Nucleosideanalogues, Novir, Oseltamivir (Tamiflu), Peginterferon alfa-2a,Penciclovir, Peramivir, Pleconaril, Podophyllotoxin, Protease inhibitor,Raltegravir, Reverse transcriptase inhibitor, Ribavirin, Rimantadine,Ritonavir, Pyramidine, Saquinavir, Sofosbuvir, Stavudine, Synergisticenhancer, Tea tree oil, Telaprevir, Tenofovir, Tenofovir disoproxil,Tipranavir, Trifluridine, Trizivir, Tromantadine, Truvada, Valaciclovir(Valtrex), Valganciclovir, Vicriviroc, Vidarabine, Viramidine,Zalcitabine, Zanamivir (Relenza), Zidovudine, and combinations thereof;and at least one pharmaceutically acceptable carrier.

The invention provides a pharmaceutical composition comprising: i) afirst therapeutic agent which is at least one an antiviral agent orpharmaceutically acceptable salt thereof; ii) a second therapeutic agentwhich at least one GCR antagonist selected from the group consisting ofPT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof; and iii) at least onepharmaceutically acceptable carrier; wherein the antiviral agent and theGCR antagonist are each present in an amount which, in combination, is atherapeutically effective amount for treating or preventing viralinfection in a patient. The invention provides a pharmaceuticalcomposition wherein the antiviral agent is selected from the groupconsisting of: Abacavir, Ziagen, Trizivir, Kivexa/Epzicom, Aciclovir,Acyclovir, Adefovir, Amantadine, Amprenavir, Ampligen, Arbidol,Atazanavir, Atripla, Balavir, Cidofovir, Combivir, Dolutegravir,Darunavir, Delavirdine, Didanosine, Docosanol, Edoxudine, Efavirenz,Emtricitabine, Enfuvirtide, Entecavir, Ecoliever, Famciclovir,Fomivirsen, Fosamprenavir, Foscarnet, Fosfonet, Ganciclovir,Ibacitabine, Imunovir, Idoxuridine, Imiquimod, Indinavir, Inosine,Integrase inhibitor, Interferon type III, Interferon type II, Interferontype I, Interferon, Lamivudine, Lopinavir, Loviride, Maraviroc,Moroxydine, Methisazone, Nelfinavir, Nevirapine, Nexavir, Nucleosideanalogues, Novir, Oseltamivir (Tamiflu), Peginterferon alfa-2a,Penciclovir, Peramivir, Pleconaril, Podophyllotoxin, Protease inhibitor,Raltegravir, Reverse transcriptase inhibitor, Ribavirin, Rimantadine,Ritonavir, Pyramidine, Saquinavir, Sofosbuvir, Stavudine, Synergisticenhancer, Tea tree oil, Telaprevir, Tenofovir, Tenofovir disoproxil,Tipranavir, Trifluridine, Trizivir, Tromantadine, Truvada, Valaciclovir(Valtrex), Valganciclovir, Vicriviroc, Vidarabine, Viramidine,Zalcitabine, Zanamivir (Relenza), Zidovudine, and combinations thereof.

The invention provides a pharmaceutical composition comprising: i) afirst therapeutic agent which is selected from the group consisting ofPT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof; ii) a second therapeuticagent which is selected from the group consisting of: cholinesteraseinhibitors, Aricept, Exelon, Razadyne, memantine, and combinationsthereof; and iii) at least one pharmaceutically acceptable carrier. Theinvention provides a pharmaceutical composition comprising: i) a firsttherapeutic agent which is at least one antiviral agent orpharmaceutically acceptable salt thereof; ii) a second therapeutic agentwhich is a GCR antagonist selected from the group consisting of PT150,PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof; and iii) at least onepharmaceutically acceptable carrier, wherein the pharmaceuticalcomposition is formulated or manufactured as a liquid, an elixir, anaerosol, a spray, a powder, a tablet, a pill, a capsule, a gel, ageltab, a nano-suspension, a nano-particle, an extended release dosageform, or a topical formulation, further wherein the antiviral agent andthe GCR antagonist are each present in an amount which, in combination,is a therapeutically effective amount for treating or preventing a viralinfection in a patient. The invention provides a pharmaceuticalcomposition wherein the antiviral agent is selected from the groupconsisting of: Abacavir, Ziagen, Trizivir, Kivexa/Epzicom, Aciclovir,Acyclovir, Adefovir, Amantadine, Amprenavir, Ampligen, Arbidol,Atazanavir, Atripla, Balavir, Cidofovir, Combivir, Dolutegravir,Darunavir, Delavirdine, Didanosine, Docosanol, Edoxudine, Efavirenz,Emtricitabine, Enfuvirtide, Entecavir, Ecoliever, Famciclovir,Fomivirsen, Fosamprenavir, Foscarnet, Fosfonet, Ganciclovir,Ibacitabine, Imunovir, Idoxuridine, Imiquimod, Indinavir, Inosine,Integrase inhibitor, Interferon type III, Interferon type II, Interferontype I, Interferon, Lamivudine, Lopinavir, Loviride, Maraviroc,Moroxydine, Methisazone, Nelfinavir, Nevirapine, Nexavir, Nucleosideanalogues, Novir, Oseltamivir (Tamiflu), Peginterferon alfa-2a,Penciclovir, Peramivir, Pleconaril, Podophyllotoxin, Protease inhibitor,Raltegravir, Reverse transcriptase inhibitor, Ribavirin, Rimantadine,Ritonavir, Pyramidine, Saquinavir, Sofosbuvir, Stavudine, Synergisticenhancer, Tea tree oil, Telaprevir, Tenofovir, Tenofovir disoproxil,Tipranavir, Trifluridine, Trizivir, Tromantadine, Truvada, Valaciclovir(Valtrex), Valganciclovir, Vicriviroc, Vidarabine, Viramidine,Zalcitabine, Zanamivir (Relenza), Zidovudine, and combinations thereof.

The invention provides a method of treating and/or preventing a viralcondition in a patient comprising: selecting a patient in need oftreating and/or preventing a viral condition; administering to thepatient at least one active agent selected from the group consisting ofPT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof; administering at least oneadditional active agent selected from the group consisting of moleculeswith potential to bind viral PS, annexin-5, anti-PS monoclonal orpolyclonal antibodies, bavituximab, wherein the viral condition is toprevent chronic latent viral infection from becoming active(reactivation), to diminish intensity of viral reactivation, to diminishlength of viral reactivation, to speed time to resolution and healing ofviral reactivation, to speed time to suppression of viral reactivation,to increase likelihood of viral eradication, and/or to diminishinfectivity of viral reactivation.

The invention provides a method wherein the viral condition is selectedfrom the group consisting of Ebola and Marburg virus (Filoviridae); RossRiver virus, chikungunya virus, Sindbis virus, eastern equineencephalitis virus (Togaviridae, Alphavirus), vesicular stomatitis virus(Rhabdoviridae, Vesiculovirus), Amapari virus, Pichindé virus, Tacaribevirus, Junin virus, Machupo virus (Arenaviridae, Mammarenavirus), WestNile virus, dengue virus, yellow fever virus (Flaviviridae, Flavivirus);human immunodeficiency virus type 1 (Retroviridae, Lentivirus); Moloneymurine leukemia virus (Retroviridae, Gammaretrovirus); influenza A virus(Orthomyxoviridae); respiratory syncytial virus (Paramyxoviridae,Pneumovirinae, Pneumovirus); vaccinia virus (Poxviridae,Chordopoxvirinae, Orthopoxvirus); herpes simplex virus type 1, herpessimplex virus type 2 (Herpesviridae, Alphaherpesvirinae, Simplexvirus);human cytomegalovirus (Herpesviridae, Betaherpesvirinae,Cytomegalovirus); Autographa californica nucleopolyhedrovirus(Baculoviridae, Alphabaculoviridae) (an insect virus); Ebola and Marburgvirus (Filoviridae); Semliki Forest virus, Ross River virus, chikungunyavirus, O'nyong-nyong virus, Sindbis virus, eastern/western/Venezuelanequine encephalitis virus (Togaviridae, Alphavirus); rubella (Germanmeasles) virus (Togaviridae, Rubivirus); rabies virus, Lagos bat virus,Mokola virus (Rhabdoviridae, Lyssavirus); Amapari virus, Pichindé virus,Tacaribe virus, Junin virus, Machupo virus, Guanarito virus, Sabiavirus, Lassa virus (Arenaviridae, Mammarenavirus); West Nile virus,dengue virus, yellow fever virus, Zika virus, Japanese encephalitisvirus, St. Louis encephalitis virus, tick-borne encephalitis virus, Omskhemorrhagic fever virus, Kyasanur Forest virus (Flaviviridae,Flavivirus); human hepatitis C virus (Flaviviridae, Hepacivirus); humanimmunodeficiency virus type 1 (Retroviridae, Lentivirus); influenza A/Bvirus (Orthomyxoviridae, the common ‘flu’ virus); respiratory syncytialvirus (Paramyxoviridae, Pneumovirinae, Pneumovirus); Hendra virus, Nipahvirus (Paramyxoviridae, Paramyxovirinae, Henipavirus); measles virus(Paramyxoviridae, Paramyxovirinae, Morbillivirus); variola major(smallpox) virus (Poxviridae, Chordopoxvirinae, Orthopoxvirus); humanhepatitis B virus (Hepadnaviridae, Orthohepadnavirus); hepatitis deltavirus (hepatitis D virus) (unassigned Family, Deltavirus); herpessimplex virus type 1, herpes simplex virus type 2 (Herpesviridae,Alphaherpesvirinae, Simplexvirus); human cytomegalovirus (Herpesviridae,Betaherpesvirinae, Cytomegalovirus)

The invention provides a method of treating and/or preventing a viralcondition in a patient comprising: selecting a patient in need oftreating and/or preventing a viral condition; administering to thepatient at least one active agent selected from the group consisting ofPT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof; administering at least oneadditional active agent selected from the group consisting of moleculeswith potential to bind viral PS, annexin-5, anti-PS monoclonal orpolyclonal antibodies, bavituximab, and/or bind to viral glucocorticoidresponse elements (GREs), retinazone, RU486 or their derivatives,wherein the at least one active agent and the at least one additionalactive agent may be used together or in sequence, to act in an additiveor synergistic fashion to prevent or treat acute viral infection, toprevent acute viral infection from becoming latent or active chronicinfection, to prevent chronic latent infection from becoming activeinfection, or to eliminate chronic latent viral infection.

The invention provides a method wherein the viral condition is selectedfrom the group consisting of Ebola and Marburg virus (Filoviridae); RossRiver virus, chikungunya virus, Sindbis virus, eastern equineencephalitis virus (Togaviridae, Alphavirus), vesicular stomatitis virus(Rhabdoviridae, Vesiculovirus), Amapari virus, Pichindé virus, Tacaribevirus, Junin virus, Machupo virus (Arenaviridae, Mammarenavirus), WestNile virus, dengue virus, yellow fever virus (Flaviviridae, Flavivirus);human immunodeficiency virus type 1 (Retroviridae, Lentivirus); Moloneymurine leukemia virus (Retroviridae, Gammaretrovirus); influenza A virus(Orthomyxoviridae); respiratory syncytial virus (Paramyxoviridae,Pneumovirinae, Pneumovirus); vaccinia virus (Poxviridae,Chordopoxvirinae, Orthopoxvirus); herpes simplex virus type 1, herpessimplex virus type 2 (Herpesviridae, Alphaherpesvirinae, Simplexvirus);human cytomegalovirus (Herpesviridae, Betaherpesvirinae,Cytomegalovirus); Autographa californica nucleopolyhedrovirus(Baculoviridae, Alphabaculoviridae) (an insect virus); Ebola and Marburgvirus (Filoviridae); Semliki Forest virus, Ross River virus, chikungunyavirus, O'nyong-nyong virus, Sindbis virus, eastern/western/Venezuelanequine encephalitis virus (Togaviridae, Alphavirus); rubella (Germanmeasles) virus (Togaviridae, Rubivirus); rabies virus, Lagos bat virus,Mokola virus (Rhabdoviridae, Lyssavirus); Amapari virus, Pichindé virus,Tacaribe virus, Junin virus, Machupo virus, Guanarito virus, Sabiavirus, Lassa virus (Arenaviridae, Mammarenavirus); West Nile virus,dengue virus, yellow fever virus, Zika virus, Japanese encephalitisvirus, St. Louis encephalitis virus, tick-borne encephalitis virus, Omskhemorrhagic fever virus, Kyasanur Forest virus (Flaviviridae,Flavivirus); human hepatitis C virus (Flaviviridae, Hepacivirus); humanimmunodeficiency virus type 1 (Retroviridae, Lentivirus); influenza A/Bvirus (Orthomyxoviridae, the common ‘flu’ virus); respiratory syncytialvirus (Paramyxoviridae, Pneumovirinae, Pneumovirus); Hendra virus, Nipahvirus (Paramyxoviridae, Paramyxovirinae, Henipavirus); measles virus(Paramyxoviridae, Paramyxovirinae, Morbillivirus); variola major(smallpox) virus (Poxviridae, Chordopoxvirinae, Orthopoxvirus); humanhepatitis B virus (Hepadnaviridae, Orthohepadnavirus); hepatitis deltavirus (hepatitis D virus) (unassigned Family, Deltavirus); herpessimplex virus type 1, herpes simplex virus type 2 (Herpesviridae,Alphaherpesvirinae, Simplexvirus); human cytomegalovirus (Herpesviridae,Betaherpesvirinae, Cytomegalovirus).

The invention provides a method of treating and/or preventing a viralcondition in a patient comprising: selecting a patient in need oftreating and/or preventing a viral condition; administering to thepatient at least one active agent selected from the group consisting ofPT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof; administering at least oneadditional active agent selected from the group consisting of moleculewith potential to bind to viral glucocorticoid response elements (GREs),retinazone, RU486 or their derivatives may be used together or insequence, to act in an additive or synergistic fashion to prevent ortreat acute viral infection, to prevent acute viral infection frombecoming latent or active chronic infection, to prevent chronic latentinfection from becoming active infection, or to eliminate chronic latentviral infection and thereby.

The invention provides a method wherein the viral condition is selectedfrom the group consisting of treat diseases caused by viral induced orassociated injuries and diseases (necrosis, inflammation, sclerosis) intissues including, but not limited to: eye (retina, sclera, lens, iris,pupil, cornea, macula, retinal blood vessels, optic nerve), ear (earcanal, bones of middle ear, tympanic membrane, Eustachian, cochlearnerve, vestibular nerve, semicircular canals, cochlea), nose (naris,vestibule, turbinates, sinuses), oral cavity and oropharynx (lips,gingiva, hard and soft palates, salivary glands, uvula, tonsils,adenoids, teeth), central nervous system and associated structures(brain, cerebrum, cerebellum, olvactory bulb, hypothalamus, reticularformation, medulla oblongata, meninges, ventricles, thalamus, pinealgland), peripheral and enteric nervous systems (autonomic nerves,sympathetic nerves, parasympathetic nerves, sensory nerves, ganglioncells, ganglia), skin (epidermis, dermis, adnexal structures, sebaceousglands, hair follicles, stratum corneum, granular cells, spinous cells,sweat glands), respiratory tract (larynx, trachea, bronchi, bronchioles,lung, alveoli, pleura), digestive tract (pharynx, esophagus, stomach,small intestine, duodenum, jejunum, ileum, colon, rectum, appendix,anus), liver (intra- and extra-hepatic bile ducts, gallbladder, liver,hepatocytes, ductules, canals of Hering), pancreas (endocrine pancreas,exocrine pancreas, pancreatic ducts, pancreatic acini), urinary tract(renal cortex, renal tubules, renal pelvis, glomeruli, ureters, urinarybladder, urethra), male genital tract (prostate, testes, scrotum,epididymis, vas deferens, glans, foreskin, corpus spongiosum, corpuscavernosum, Cowper's gland), female genital tract (ovary, fimbria,fallopian tubes, uterus, endometrium, endocervix, endocervical glands,cervix, ectocervix, vagina, labia, placenta), endocrine system (pinealglands, pituitary gland, thyroid gland, parathyroid glands, adrenalglands, adrenal cortex, adrenal medulla), cardiovascular system (heart,pericardium, myocardium, endocardium, atria, ventricles, coronaryarteries, tricuspid valve, aortic valve, mitral valve, pulmonic valve,aorta, arteries, arterioles, capillaries, venules, veins, inferior venacava, superior vena cava, pulmonary artery, pulmonic vein),musculoskeletal system (bones, tendons, ligaments, skeletal muscle,smooth muscle, fascia) and blood (platelets, red blood cells, whiteblood cells, and all their precursors, and bone marrow).

The invention provides a method of treating and/or preventing a viralcondition in a patient comprising: selecting a patient in need oftreating and/or preventing a viral condition; administering to thepatient at least one active agent selected from the group consisting ofPT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof; administering at least oneadditional active agent selected from the group consisting of moleculeswith potential to bind viral PS, annexin-5, anti-PS monoclonal orpolyclonal antibodies, bavituximab may be used together or in sequence,to act in an additive or synergistic fashion to prevent or treat acuteviral infection, to prevent acute viral infection from becoming latentor active chronic infection, to prevent chronic latent infection frombecoming active infection, or to eliminate chronic latent viralinfection and thereby treat diseases caused by viral induced orassociated injuries and diseases (necrosis, inflammation, sclerosis) intissues including, but not limited to: eye (retina, sclera, lens, iris,pupil, cornea, macula, retinal blood vessels, optic nerve), ear (earcanal, bones of middle ear, tympanic membrane, Eustachian, cochlearnerve, vestibular nerve, semicircular canals, cochlea), nose (naris,vestibule, turbinates, sinuses), oral cavity and oropharynx (lips,gingiva, hard and soft palates, salivary glands, uvula, tonsils,adenoids, teeth), central nervous system and associated structures(brain, cerebrum, cerebellum, olvactory bulb, hypothalamus, reticularformation, medulla oblongata, meninges, ventricles, thalamus, pinealgland), peripheral and enteric nervous systems (autonomic nerves,sympathetic nerves, parasympathetic nerves, sensory nerves, ganglioncells, ganglia), skin (epidermis, dermis, adnexal structures, sebaceousglands, hair follicles, stratum corneum, granular cells, spinous cells,sweat glands), respiratory tract (larynx, trachea, bronchi, bronchioles,lung, alveoli, pleura), digestive tract (pharynx, esophagus, stomach,small intestine, duodenum, jejunum, ileum, colon, rectum, appendix,anus), liver (intra- and extra-hepatic bile ducts, gallbladder, liver,hepatocytes, ductules, canals of Hering), pancreas (endocrine pancreas,exocrine pancreas, pancreatic ducts, pancreatic acini), urinary tract(renal cortex, renal tubules, renal pelvis, glomeruli, ureters, urinarybladder, urethra), male genital tract (prostate, testes, scrotum,epididymis, vas deferens, glans, foreskin, corpus spongiosum, corpuscavernosum, Cowper's gland), female genital tract (ovary, fimbria,fallopian tubes, uterus, endometrium, endocervix, endocervical glands,cervix, ectocervix, vagina, labia, placenta), endocrine system (pinealglands, pituitary gland, thyroid gland, parathyroid glands, adrenalglands, adrenal cortex, adrenal medulla), cardiovascular system (heart,pericardium, myocardium, endocardium, atria, ventricles, coronaryarteries, tricuspid valve, aortic valve, mitral valve, pulmonic valve,aorta, arteries, arterioles, capillaries, venules, veins, inferior venacava, superior vena cava, pulmonary artery, pulmonic vein),musculoskeletal system (bones, tendons, ligaments, skeletal muscle,smooth muscle, fascia) and blood (platelets, red blood cells, whiteblood cells, and all their precursors, and bone marrow).

The invention provides a method of treating and/or preventing a viralcondition in a patient comprising: selecting a patient in need oftreating and/or preventing a viral condition; administering to thepatient at least one active agent selected from the group consisting ofPT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof; administering at least oneadditional active agent selected from the group consisting of moleculeswith potential to bind viral PS, annexin-5, anti-PS monoclonal orpolyclonal antibodies, bavituximab, and/or bind to viral glucocorticoidresponse elements (GREs), retinazone, RU486 or their derivatives,wherein the active agents may be used together or in sequence, to act inan additive or synergistic fashion to prevent or treat acute viralinfection, to prevent acute viral infection from becoming latent oractive chronic infection, to prevent chronic latent infection frombecoming active infection, or to eliminate chronic latent viralinfection and thereby treat diseases caused by viral induced orassociated injuries and diseases (necrosis, inflammation, sclerosis) intissues including, but not limited to: eye (retina, sclera, lens, iris,pupil, cornea, macula, retinal blood vessels, optic nerve), ear (earcanal, bones of middle ear, tympanic membrane, Eustachian, cochlearnerve, vestibular nerve, semicircular canals, cochlea), nose (naris,vestibule, turbinates, sinuses), oral cavity and oropharynx (lips,gingiva, hard and soft palates, salivary glands, uvula, tonsils,adenoids, teeth), central nervous system and associated structures(brain, cerebrum, cerebellum, olvactory bulb, hypothalamus, reticularformation, medulla oblongata, meninges, ventricles, thalamus, pinealgland), peripheral and enteric nervous systems (autonomic nerves,sympathetic nerves, parasympathetic nerves, sensory nerves, ganglioncells, ganglia), skin (epidermis, dermis, adnexal structures, sebaceousglands, hair follicles, stratum corneum, granular cells, spinous cells,sweat glands), respiratory tract (larynx, trachea, bronchi, bronchioles,lung, alveoli, pleura), digestive tract (pharynx, esophagus, stomach,small intestine, duodenum, jejunum, ileum, colon, rectum, appendix,anus), liver (intra- and extra-hepatic bile ducts, gallbladder, liver,hepatocytes, ductules, canals of Hering), pancreas (endocrine pancreas,exocrine pancreas, pancreatic ducts, pancreatic acini), urinary tract(renal cortex, renal tubules, renal pelvis, glomeruli, ureters, urinarybladder, urethra), male genital tract (prostate, testes, scrotum,epididymis, vas deferens, glans, foreskin, corpus spongiosum, corpuscavernosum, Cowper's gland), female genital tract (ovary, fimbria,fallopian tubes, uterus, endometrium, endocervix, endocervical glands,cervix, ectocervix, vagina, labia, placenta), endocrine system (pinealglands, pituitary gland, thyroid gland, parathyroid glands, adrenalglands, adrenal cortex, adrenal medulla), cardiovascular system (heart,pericardium, myocardium, endocardium, atria, ventricles, coronaryarteries, tricuspid valve, aortic valve, mitral valve, pulmonic valve,aorta, arteries, arterioles, capillaries, venules, veins, inferior venacava, superior vena cava, pulmonary artery, pulmonic vein),musculoskeletal system (bones, tendons, ligaments, skeletal muscle,smooth muscle, fascia) and blood (platelets, red blood cells, whiteblood cells, and all their precursors, and bone marrow).

The invention provides a method of treating and/or preventing a viralcondition in a patient comprising: selecting a patient in need oftreating and/or preventing a viral condition; administering to thepatient at least one active agent selected from the group consisting ofPT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof; administering at least oneadditional active agent selected from the group consisting of moleculeswith potential to bind viral PS annexin-5, anti-PS monoclonal orpolyclonal antibodies, bavituximab and/or bind to viral glucocorticoidresponse elements (GREs), retinazone, RU486 or their derivatives) andone or more anti-viral medications including, but not limited to cellentry inhibitors, uncoating inhibitors, reverse transcriptaseinhibitors, integrase inhibitors, transcription inhibitors, translation(antisense) inhibitors, translation (ribozyme) inhibitors, preinprocessing and targeting inhibitors, protease inhibitors, assemblyinhibitors, release phase inhibitos, immunosystem modulators andvaccines may be used together or in sequence, to act in an additive orsynergistic fashion to prevent or treat acute viral infection, toprevent acute viral infection from becoming latent or active chronicinfection, to prevent chronic latent infection from becoming activeinfection, or to eliminate chronic latent viral infection and therebytreat diseases caused by viral induced or associated injuries anddiseases (necrosis, inflammation, sclerosis) in tissues including, butnot limited to: eye (retina, sclera, lens, iris, pupil, cornea, macula,retinal blood vessels, optic nerve), ear (ear canal, bones of middleear, tympanic membrane, Eustachian, cochlear nerve, vestibular nerve,semicircular canals, cochlea), nose (naris, vestibule, turbinates,sinuses), oral cavity and oropharynx (lips, gingiva, hard and softpalates, salivary glands, uvula, tonsils, adenoids, teeth), centralnervous system and associated structures (brain, cerebrum, cerebellum,olvactory bulb, hypothalamus, reticular formation, medulla oblongata,meninges, ventricles, thalamus, pineal gland), peripheral and entericnervous systems (autonomic nerves, sympathetic nerves, parasympatheticnerves, sensory nerves, ganglion cells, ganglia), skin (epidermis,dermis, adnexal structures, sebaceous glands, hair follicles, stratumcorneum, granular cells, spinous cells, sweat glands), respiratory tract(larynx, trachea, bronchi, bronchioles, lung, alveoli, pleura),digestive tract (pharynx, esophagus, stomach, small intestine, duodenum,jejunum, ileum, colon, rectum, appendix, anus), liver (intra- andextra-hepatic bile ducts, gallbladder, liver, hepatocytes, ductules,canals of Hering), pancreas (endocrine pancreas, exocrine pancreas,pancreatic ducts, pancreatic acini), urinary tract (renal cortex, renaltubules, renal pelvis, glomeruli, ureters, urinary bladder, urethra),male genital tract (prostate, testes, scrotum, epididymis, vas deferens,glans, foreskin, corpus spongiosum, corpus cavernosum, Cowper's gland),female genital tract (ovary, fimbria, fallopian tubes, uterus,endometrium, endocervix, endocervical glands, cervix, ectocervix,vagina, labia, placenta), endocrine system (pineal glands, pituitarygland, thyroid gland, parathyroid glands, adrenal glands, adrenalcortex, adrenal medulla), cardiovascular system (heart, pericardium,myocardium, endocardium, atria, ventricles, coronary arteries, tricuspidvalve, aortic valve, mitral valve, pulmonic valve, aorta, arteries,arterioles, capillaries, venules, veins, inferior vena cava, superiorvena cava, pulmonary artery, pulmonic vein), musculoskeletal system(bones, tendons, ligaments, skeletal muscle, smooth muscle, fascia) andblood (platelets, red blood cells, white blood cells, and all theirprecursors, and bone marrow).

The invention provides a method of treating immunocompetent patients whohave mycobacterial infection comprising: selecting a patient in need oftreating and/or preventing the condition; administering to the patientat least one active agent selected from the group consisting of PT150,PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof; wherein the mycobacterialinfection is mycobacterium tuberculosis (MTB) and leprosy, identified byroutine screening in the absence of clinical signs or symptoms ofmycobacterial infection, identified by serologic studies, identified incultured tissue, identified on histochemical or immunostains in tissuebiopsy or resection specimens, or presenting with clinical signs orsymptoms of mycobacterial infection, in order to antagonize physiologiccortisol-mediated suppression of the immune system and therebyfacilitating immune-clearance of infection.

The invention provides a method of treating immunocompetent patients whohave mycobacterial infection comprising: selecting a patient in need oftreating and/or preventing the condition; administering to the patientat least one active agent selected from the group consisting of PT150,PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof, wherein the mycobacterialinfection including, but not limited to mycobacterium tuberculosis(MTB), leprosy, mycobacterium avium intracellulare (MAI), mycobacteriumkansasii, mycobacterium fortuitum, identified by routine screening inthe absence of clinical signs or symptoms of mycobacterial infection,identified by serologic studies, identified in cultured tissue,identified on histochemical or immunostains in tissue biopsy orresection specimens, or presenting with clinical signs or symptoms ofmycobacterial infection, in order to antagonize physiologiccortisol-mediated suppression of the immune system and therebyfacilitating immune-clearance of infection.

The invention provides a pharmaceutical composition comprising: at leastone glucocorticoid antagonist, selected from the group consisting ofPT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof; optionally, antibiotics withanti-mycobacterial infection (including, but not limited to rifamycins,isoniazid, pyrazinamide, ethambutol, fuloroquinolones, aminoglycosides,ethionamide, cycloserine, capreomycin, dapsone, clofazimine,minocycline, clarithromycin, macrolides; at least one pharmaceuticallyacceptable excipient.

The invention provides a method of treating and/or preventing addictionin a patient comprising: selecting a patient in need of treating and/orpreventing addiction; continuously or intermittently administering tothe patient at least one active agent selected from the group consistingof PT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof, wherein the at least oneactive agent prevents triggering of Pavlovian sign-tracking and therebydiminish or eliminate likely relapse in response to cues originallyassociated with the use of the addictive compound.

The invention provides a method of treating and/or preventing addictionin a patient comprising: selecting a patient in need of treating and/orpreventing the addiction; continuously or intermittently administeringto the patient at least one active agent selected from the groupconsisting of PT150, PT155, PT156, PT157, PT158, TCY1, combinationsthereof, and pharmaceutically acceptable salts thereof, wherein the atleast one active agent prevents development of Pavlovian sign-trackingand thereby diminishing or prevent progression from regular(recreational or medicinal) use to physical addiction.

The invention provides a method of treating or preventing Addison'sDisease in a patient, the method comprising: selecting a patient in needof treating and/or preventing Addison's Disease; administering to thepatient the pharmaceutical composition of claim 32, as a way ofpreventing or minimizing development of Addisonian symptoms fromongoing, high level systemic GCR blockade of endogenous, physiologiccortisol, thereby treating and/or preventing Addison's Disease. Theinvention provides a method of treating and/or preventing reactivationof viral infection in a patient comprising: selecting a patient in needof treating and/or preventing viral infection; administering to thepatient prior to or receiving therapeutic doses of glucocorticoids atleast one active agent selected from the group consisting of PT150,PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof.

The invention provides a method of treating and/or preventingreactivation of latent viral infection in a patient comprising:selecting a patient in need of treating and/or preventing latent viralinfection; administering to the patient prior to or receivingtherapeutic doses of glucocorticoids at least one active agent selectedfrom the group consisting of PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof,wherein the administration diminish intensity of viral reactivation, todiminish length of viral reactivation, to speed time to resolution andhealing of viral reactivation, to speed time to suppression of viralreactivation, to increase likelihood of viral eradication, and/or todiminish infectivity of viral reactivation.

The invention provides a method of treating and/or preventingreactivation of viral infection in a patient comprising: selecting apatient in need of treating and/or preventing viral infection;administering to the patient prior to or receiving therapeutic doses ofglucocorticoids at least one active agent selected from the groupconsisting of PT150, PT155, PT156, PT157, PT158, TCY1, combinationsthereof, and pharmaceutically acceptable salts thereof, wherein theadministration diminishes intensity of viral infection, to diminishlength of viral infection, to speed time to resolution and healing ofviral infection, to speed time to suppression of viral infection, toincrease likelihood of viral eradication, and/or to diminish infectivityof viral infection.

The invention provides a method of treating and/or preventingreactivation of viral infection in a patient comprising: selecting apatient in need of treating and/or preventing viral infection;administering to the patient prior to or during travel to environmentsin which viruses are endemic at least one active agent selected from thegroup consisting of PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof.

The invention provides a method of treating and/or preventing dementiain a patient comprising: selecting a patient in need of treating and/orpreventing dementia; administering to the patient at least one activeagent selected from the group consisting of PT150, PT155, PT156, PT157,PT158, TCY1, combinations thereof, and pharmaceutically acceptable saltsthereof; administering to the patient at least one additionaltherapeutic agent which is selected from the group consisting ofcholinesterase inhibitors, Aricept, Exelon, Razadyne, and memantine,thereby treating and/or preventing dementia in the patient.

The invention provides a pharmaceutical composition made by combining atleast one antiviral agent or pharmaceutically acceptable salt thereof,at least one a GCR antagonist selected from the group consisting ofPT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof, and a pharmaceuticallyacceptable carrier. The invention provides a pharmaceutical activesubstance combination comprising: i) a first therapeutic agent which isat least one antiviral agent or pharmaceutically acceptable saltthereof; ii) a second therapeutic agent which is a GCR antagonistselected from the group consisting of PT150, PT155, PT156, PT157, PT158,TCY1, combinations thereof, and pharmaceutically acceptable saltsthereof, as a combination product for simultaneous, separate, orsequential use. The invention provides a pharmaceutical dosage formcomprising: i) a first therapeutic agent which is at least one antiviralagent or pharmaceutically acceptable salt thereof; ii) a secondtherapeutic agent which is a GCR antagonist selected from the groupconsisting of PT150, PT155, PT156, PT157, PT158, TCY1, combinationsthereof, and pharmaceutically acceptable salts thereof, wherein thefirst and second agents are in multiple separated dosage units or in asingle dosage unit of a combination of the therapeutic agents.

The invention provides a kit for the treatment, amelioration orprevention of a viral infection condition in a patient in need of suchtreatment comprising: (a) the pharmaceutical composition herein; and (b)at least one blister package; a lidded blister; a blister card orpacket; a clamshell; an intravenous (IV) package, IV packette or IVcontainer; a tray or a shrink wrap comprising the pharmaceuticalcomposition of (a) and instructions for use of the pharmaceuticalcomposition. The invention provides a pharmaceutical packaging systemcomprising: i) a first therapeutic agent which is an antiviral agent, orpharmaceutically acceptable salts thereof; ii) a second therapeuticagent which is a GCR antagonist, or pharmaceutically acceptable saltsthereof, wherein the means for containing said therapeutic dosages isselected from the group consisting of the first and second agents are ina single dosage form; the first and second agents are packaged togetherin a single package or packette; the first and second agents arepackaged separately in a plurality of packages or packettes; a blisterpacket; a lidded blister; or blister card or packets; a shrink wrap, andwith both drugs released upon opening of the single package or packette;a plurality of packages or packettes; blister packet; lidded blister orblister card or packets; or shrink wrap; a blister pack; a container;and a device, and wherein the dosages are separated from each otherwithin the pharmaceutical packaging system.

The invention provides a process for making a pharmaceutical compositioncomprising combining at least one antiviral agent or pharmaceuticallyacceptable salts thereof, at least one GCR antagonist selected from thegroup consisting of PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof, andat least one pharmaceutically acceptable carrier.

The invention provides a method of treating or preventing viralinfection in a patient, comprising: selecting a patient in need oftreating or preventing viral infection; administering to the patient acomposition comprising: i) a first therapeutic agent which is aantiviral agent, or pharmaceutically acceptable salts thereof; ii) asecond therapeutic agent which is a GCR antagonist or pharmaceuticallyacceptable salts thereof; and iii) at least one a pharmaceuticallyacceptable carrier, wherein the antiviral agent and the GCR antagonistare each present in an amount which, in combination, is atherapeutically effective amount for treating or preventing viralinfection in a patient.

The invention provides a method wherein the antiviral agent is selectedfrom the group consisting of: Abacavir, Ziagen, Trizivir,Kivexa/Epzicom, Aciclovir, Acyclovir, Adefovir, Amantadine, Amprenavir,Ampligen, Arbidol, Atazanavir, Atripla, Balavir, Cidofovir, Combivir,Dolutegravir, Darunavir, Delavirdine, Didanosine, Docosanol, Edoxudine,Efavirenz, Emtricitabine, Enfuvirtide, Entecavir, Ecoliever,Famciclovir, Fomivirsen, Fosamprenavir, Foscarnet, Fosfonet,Ganciclovir, Ibacitabine, Imunovir, Idoxuridine, Imiquimod, Indinavir,Inosine, Integrase inhibitor, Interferon type III, Interferon type II,Interferon type I, Interferon, Lamivudine, Lopinavir, Loviride,Maraviroc, Moroxydine, Methisazone, Nelfinavir, Nevirapine, Nexavir,Nucleoside analogues, Novir, Oseltamivir (Tamiflu). Peginterferonalfa-2a, Penciclovir, Peramivir, Pleconaril, Podophyllotoxin, Proteaseinhibitor, Raltegravir, Reverse transcriptase inhibitor, Ribavirin,Rimantadine, Ritonavir, Pyramidine, Saquinavir, Sofosbuvir, Stavudine,Synergistic enhancer, Tea tree oil, Telaprevir, Tenofovir, Tenofovirdisoproxil, Tipranavir, Trifluridine, Trizivir, Tromantadine, Truvada,Valaciclovir (Valtrex), Valganciclovir, Vicriviroc, Vidarabine,Viramidine, Zalcitabine, Zanamivir, Zidovudine, and combinationsthereof.

The invention provides a method wherein the GCR antagonist is selectedfrom the group consisting of is selected from the group consisting ofPT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof.

The present invention relates to a method for detecting the presence ofa biological substance in a test sample, comprising the steps ofproviding a test sample consisting of, for example, saliva, or a bodilyfluid sample from a subject with, for example, a lollipop-like apparatusincluding a stem integrated with the base and a head integrated with thestem. The stem head including a receptor of a sponge like carrier toensure a high void volume to absorb sufficient saliva, oral fluid or abodily fluid sample.

Combining the test sample with a buffering system (Reagent 1) containingviscosity controllers and stabilizers into a reaction vessel, mixing thesolution well, and expressing all the liquid from the sample carrierapparatus into Reagent 1 in the reaction vessel and discarding. Readingthe reaction vessel with sample and buffer for a fluorescencepolarization blank and then combining the test sample and buffer mixturewith a fluorescence-labeled ligand (Reagent 2) to said biologicalsubstance in the reaction vessel, mix solution well, to produce an assaysolution. Furthermore, Reagent 2 may be delivered to the reaction vesselwithout further dilution volume of the assay solution.

The invention provides a method for screening a patient for a diseasestate suitable for GCR (glucocorticoid receptor) antagonist therapy,comprising the steps of: a) obtaining a test sample from the patient,optionally at a predetermined time, using a test sample collection unit;b) combining said test sample with a buffering system to form a mixturein a reaction unit; c) measuring a parameter of the mixture to determinea blank measurement; d) combining said test sample and buffer mixturewith a labeled ligand which binds cortisol, wherein the labeled ligandis provided in a label unit, in the reaction unit to produce an assaysolution; or combining said test sample and buffer mixture anddelivering it to a carrier containing a labeled ligand which bindscortisol, wherein the labeled ligand is provided in a label unit, in thereaction unit to produce an assay immobilized complex; e) measuring aparameter of said assay solution or complex; f) comparing themeasurement of the assay solution relative to the blank measurement; g)determining the patient's circulating cortisol levels based on thechange of the measurement; and h) comparing the patient's measuredcortisol levels to a predetermined reference range cortisol levels,wherein when the level of cortisol is elevated relative to thepredetermined reference range, then the patient has a disease statewhich involves elevated cortisol, and thus has a disease state which isa potential candidate for GCR antagonist or active agent therapy. Theinvention further provides a method wherein the patient's test sample isselected from the group consisting of saliva, blood, plasma, serum,urine, other bodily fluids, and combinations thereof. The inventionfurther provides a method wherein the sample is obtained from thepatient over more than one time, and the predetermined time is selectedfrom the group consisting of morning, noon, and evening. The inventionfurther provides a method wherein the sample is obtained from thepatient over consecutive days. The invention further provides a methodwherein the method is to determine the circadian cycle of the cortisollevels in the patient, and the predetermined time is selected from thegroup consisting of about hourly, every 4 hours, every 6 hours, every 8hours, and every 12 hours. The invention further provides a methodwherein the sample is obtained from the patient over consecutive days.The invention further provides a method wherein the predeterminedreference range is a medically standard reference range. The inventionfurther provides a method wherein the predetermined reference range isthe patient's previously measured level. The invention further providesa method wherein the ligand is detectably labeled with a moiety selectedfrom the group consisting of a radioisotope, a fluorophore, a quencherof fluorescence, an enzyme, an affinity tag, and an epitope tag. Theinvention further provides a method wherein said measuring of saidparameter of said mixture and said assay solution is performed using amethod selected from spectroscopic, photochemical, radiochemical,biochemical, enzymatic, immunochemical, chemical label displacement,surface plasmon resonance, fluorescence resonance energy transfer,fluorescence quenching, lateral flow, and fluorescence polarizationmeans. The invention further provides a method further comprising thesteps of i) determining a patient's elevated cortisol level; and j)providing a therapeutic for such elevated cortisol level, wherein thetherapeutic comprises GCR antagonist or active agent therapy. Theinvention further provides a method wherein the patient has changedpatterns of cortisol levels that have been observed in connection withabnormal Adrenocorticotropic hormone (ACTH) levels. The inventionfurther provides a method wherein the patient has non-normal cortisollevels produced by the adrenal cortex or disordered circadian rhythms,as a method for selecting subjects for GCR antagonist or active agenttherapy wherein the patient has cortisol levels selected from the groupconsisting abnormally high cortisol levels but maintained circadianrhythm, over responsiveness to normal levels, and high night timecortisol levels as a feature of disrupted circadian rhythm. Theinvention further provides a method wherein the disease state isselected from the group consisting of cancer, clinical depression,psychological stress, and physiological stressors such as hypoglycemia,illness, fever, trauma, surgery, fear, pain, physical exertion, ortemperature extremes.

The invention provides a method for monitoring changes in cortisollevels in response to treatment, in patients who have non-normalcortisol levels produced by the adrenal cortex, comprising: a) obtaininga test sample from the patient, optionally at a predetermined time,using a test sample collection unit; b) combining said test sample witha buffering system to form a mixture in a reaction unit; c) measuring aparameter of the mixture to determine a blank measurement; d) combiningsaid test sample and buffer mixture with a labeled ligand which bindscortisol, wherein the labeled ligand is provided in a label unit, in thereaction unit to produce an assay solution; or combining said testsample and buffer mixture and delivering it to a carrier containing alabeled ligand which binds cortisol, wherein the labeled ligand isprovided in a label unit, in the reaction unit to produce an assayimmobilized complex; e) measuring a parameter of said assay solution orcomplex; f) comparing the measurement of the assay solution relative tothe blank measurement; g) determining the patient's circulating cortisollevels based on the change of the measurement; h) administering a GCRantagonist; i) repeating steps a) to f) after the therapy has beenadministered; and j) determining the patient's circulating cortisollevels post-therapy, wherein when the cortisol levels change in responseto treatment to indicate responsiveness to the GCR antagonist. Theinvention further provides a method wherein the patient's test sample isselected from the group consisting of saliva, blood, plasma, serum,urine, other bodily fluids, and combinations thereof. The inventionfurther provides a method wherein the sample is obtained from thepatient over more than one day, and the predetermined time is selectedfrom the group consisting of morning, noon, and evening. The inventionfurther provides a method wherein the sample is obtained from thepatient over consecutive days. The invention further provides a methodwherein the method is to determine the circadian cycle of the cortisollevels in the patient, and the predetermined time is selected from thegroup consisting of hourly, every 4 hours, every 6 hours, every 8 hours,and every 12 hours. The invention further provides a method wherein thesample is obtained from the patient over consecutive days. The inventionfurther provides a method wherein the predetermined reference range is amedically standard reference range. The invention further provides amethod wherein the predetermined reference range is the patient'spreviously measured level. The invention further provides a methodwherein the ligand is detectably labeled with a moiety selected from thegroup consisting of a radioisotope, a fluorophore, a quencher offluorescence, an enzyme, an affinity tag, and an epitope tag. Theinvention further provides a method wherein said measuring of saidparameter of said mixture and said assay solution is performed using amethod selected from spectroscopic, photochemical, radiochemical,biochemical, enzymatic, immunochemical, chemical label displacement,surface plasmon resonance, fluorescence resonance energy transfer,fluorescence quenching, lateral flow, and fluorescence polarizationmeans.

The invention provides a method for monitoring changes in cortisollevels in response to treatment and adjusting the treatment in responseto these changes in a patient who has non-normal cortisol levelsproduced by the adrenal cortex, comprising: a) obtaining a test samplefrom the patient, optionally at a predetermined time, using a testsample collection unit; b) combining said test sample with a bufferingsystem to form a mixture in a reaction unit; c) measuring a parameter ofthe mixture to determine a blank measurement; d) combining said testsample and buffer mixture with a labeled ligand which binds cortisol,wherein the labeled ligand is provided in a label unit, in the reactionunit to produce an assay solution; or combining said test sample andbuffer mixture and delivering it to a carrier containing a labeledligand which binds cortisol, wherein the labeled ligand is provided in alabel unit, in the reaction unit to produce an assay immobilizedcomplex; e) measuring a parameter of said assay solution or complex; f)comparing the measurement of the assay solution relative to the blankmeasurement; g) determining the patient's circulating cortisol levelsbased on the change of the measurement; h) administering a GCRantagonist; i) repeating steps a) to f) after the therapy has beenadministered; j) determining the patient's circulating cortisol levelspost-therapy; and k) adjusting the GCR antagonist or active agenttherapy in response to changes in the patient's cortisol levelspost-therapy, wherein the adjustment in GCR antagonist or active agenttherapy is to enhance therapeutic efficacy.

The invention further provides a method wherein the patient's testsample is selected from the group consisting of saliva, blood, plasma,serum, urine, other bodily fluids, and combinations thereof. Theinvention further provides a method wherein the sample is obtained fromthe patient over more than one time, and the predetermined time isselected from the group consisting of morning, noon, and evening. Theinvention further provides a method wherein the sample is obtained fromthe patient over consecutive days. The invention further provides amethod wherein the method is to determine the circadian cycle of thecortisol levels in the patient, and the predetermined time is selectedfrom the group consisting of hourly, every 4 hours, every 6 hours, every8 hours, and every 12 hours. The invention further provides a methodwherein the sample is obtained from the patient over consecutive days.The invention further provides a method wherein the predeterminedreference range is a medically standard reference range. The inventionfurther provides a method wherein the predetermined reference range isthe patient's previously measured level. The invention further providesa method wherein the ligand is detectably labeled with a moiety selectedfrom the group consisting of a radioisotope, a fluorophore, a quencherof fluorescence, an enzyme, an affinity tag, and an epitope tag. Theinvention further provides a method wherein said measuring of saidparameter of said mixture and said assay solution is performed using amethod selected from spectroscopic, photochemical, radiochemical,biochemical, enzymatic, immunochemical, chemical label displacement,surface plasmon resonance, fluorescence resonance energy transfer,fluorescence quenching, lateral flow, and fluorescence polarizationmeans. The invention further provides a method wherein the GCRantagonist is selected from the group consisting of compounds which areselective for GCR, compounds which non-specifically bind steroid hormonereceptors, and compounds which cross-react to both GCR and other steroidhormone receptors. The invention further provides a method wherein adecision to adjust the GCR antagonist or active agent therapy inresponse to changes in the cortisol levels, post-therapy, is made by amedical professional. The invention further provides a method furthercomprising the step of monitoring changes in biomarker expression usinga nucleic acid microarray. The invention further provides a methodwherein in the patients having normal baseline cortisol at the start oftreatment, and changing cortisol levels during treatment indicateresponsiveness to the GCR antagonist. The invention further provides amethod wherein the combined system of salivary cortisol quantificationas an enabling device for its paired GCR antagonist will identifypatients for whom GCR antagonism has a likely benefit. The inventionfurther provides a method wherein the GCR antagonist is selected fromthe group consisting of PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof. Theinvention further provides a method wherein the buffering systemcomprises additional components selected form the group consisting ofviscosity controllers, stabilizers, and combinations thereof. Theinvention further provides a method wherein the fluorescence-labeledligand which binds cortisol is selected from the group consisting of anaptamer, an antibody, an antibody fragment, a receptor, a receptorfragment, a binding polypeptide, a binding peptide, and combinationsthereof. The invention further provides a method wherein the test sampleis collected from the patient with a lollipop-like apparatus, includinga stem integrated with the base and a head integrated with the stem, andfurther wherein the stem head including a receptor of a sponge likecarrier to ensure a high void volume to absorb sufficient sample.

The invention provides a method of treating major depressive disorder ina patient in need thereof by determining whether the patient has majordepressive disorder suitable for GCR (glucocorticoid receptor)antagonist therapy, comprising the steps of: a) obtaining a test samplefrom the patient, optionally at a predetermined time, using a testsample collection unit; b) combining said test sample with a bufferingsystem to form a mixture in a reaction unit; c) measuring a parameter ofthe mixture to determine a blank measurement; d) combining said testsample and buffer mixture with a labeled ligand which binds cortisol,wherein the labeled ligand is provided in a label unit, in the reactionunit to produce an assay solution; or combining said test sample andbuffer mixture and delivering it to a carrier containing a labeledligand which binds cortisol, wherein the labeled ligand is provided in alabel unit, in the reaction unit to produce an assay immobilizedcomplex; e) measuring a parameter of said assay solution or complex; f)comparing the measurement of the assay solution relative to the blankmeasurement; g) determining the patient's circulating cortisol levelsbased on the change of the measurement; h) comparing the measuredcortisol levels to a predetermined reference range cortisol levels,wherein when the level of cortisol is elevated relative to the apredetermined reference range, then the patient has major depressivedisorder which involves elevated cortisol, and thus has major depressivedisorder suitable for GCR (glucocorticoid receptor) antagonist therapy;and i) when the patient has major depressive disorder suitable for GCRantagonist or active agent therapy, a administering at least one GCRantagonist. The invention further provides a method wherein thepatient's test sample is selected from the group consisting of saliva,blood, plasma, serum, urine, other bodily fluids, and combinationsthereof. The invention further provides a method wherein the sample isobtained from the patient over more than one time, and the predeterminedtime is selected from the group consisting of morning, noon, andevening. The invention further provides a method wherein the sample isobtained from the patient over consecutive days. The invention furtherprovides a method wherein the method is to determine the circadian cycleof the cortisol levels in the patient, and the predetermined time isselected from the group consisting of hourly, every 4 hours, every 6hours, every 8 hours, and every 12 hours. The invention further providesa method wherein the sample is obtained from the patient overconsecutive days. The invention further provides a method wherein thepredetermined reference range is a medically standard reference range.The invention further provides a method wherein the predeterminedreference range is the patient's previously measured level. Theinvention further provides a method wherein the ligand is detectablylabeled with a moiety selected from the group consisting of aradioisotope, a fluorophore, a quencher of fluorescence, an enzyme, anaffinity tag, and an epitope tag. The invention further provides amethod wherein said measuring of said parameter of said mixture and saidassay solution is performed using a method selected from spectroscopic,photochemical, radiochemical, biochemical, enzymatic, immunochemical,chemical label displacement, surface plasmon resonance, fluorescenceresonance energy transfer, fluorescence quenching, lateral flow, andfluorescence polarization means. The invention further provides a methodwherein the GCR (glucocorticoid receptor) antagonist is selected fromthe group consisting of PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof.

The invention provides a method of treating psychotic depression in apatient in need thereof by determining whether the patient has psychoticdepression suitable for GCR (glucocorticoid receptor) antagonisttherapy, comprising the steps of a) obtaining a test sample from thepatient, optionally at a predetermined time using a test samplecollection unit; b) combining said test sample with a buffering systemto form a mixture in a reaction unit; c) measuring a parameter of themixture to determine a blank measurement; d) combining said test sampleand buffer mixture with a labeled ligand which binds cortisol, whereinthe labeled ligand is provided in a label unit, in the reaction unit toproduce an assay solution; or combining said test sample and buffermixture and delivering it to a carrier containing a labeled ligand whichbinds cortisol, wherein the labeled ligand is provided in a label unit,in the reaction unit to produce an assay immobilized complex; e)measuring a parameter of said assay solution or complex; f) comparingthe measurement of the assay solution relative to the blank measurement;g) determining the patient's circulating cortisol levels based on thechange of the measurement; h) comparing the measured cortisol levels toa predetermined reference range cortisol levels, wherein when the levelof cortisol is elevated relative to the predetermined reference range,then the patient has psychotic depression which involves elevatedcortisol, and thus has psychotic depression suitable for GCR antagonistor active agent therapy; and i) when the patient has psychoticdepression suitable for GCR antagonist or active agent therapy,administering at least one GCR antagonist. The invention furtherprovides a method wherein the patient's test sample is selected from thegroup consisting of saliva, blood, plasma, serum, urine, other bodilyfluids, and combinations thereof. The invention further provides amethod wherein the sample is obtained from the patient over more thanone time, and the predetermined time is selected from the groupconsisting of morning, noon, and evening. The invention further providesa method wherein the sample is obtained from the patient overconsecutive days. The invention further provides a method wherein themethod is to determine the circadian cycle of the cortisol levels in thepatient, and the predetermined time is selected from the groupconsisting of hourly, every 4 hours, every 6 hours, every 8 hours, andevery 12 hours. The invention further provides a method wherein thesample is obtained from the patient over consecutive days. The inventionfurther provides a method wherein the predetermined reference range is amedically standard reference range. The invention further provides amethod wherein the predetermined reference range is the patient'spreviously measured level. The invention further provides a methodwherein the ligand is detectably labeled with a moiety selected from thegroup consisting of a radioisotope, a fluorophore, a quencher offluorescence, an enzyme, an affinity tag, and an epitope tag. Theinvention further provides a method wherein said measuring of saidparameter of said mixture and said assay solution is performed using amethod selected from spectroscopic, photochemical, radiochemical,biochemical, enzymatic, immunochemical, chemical label displacement,surface plasmon resonance, fluorescence resonance energy transfer,fluorescence quenching, lateral flow, and fluorescence polarizationmeans. The invention further provides a method wherein the GCRantagonist is selected from the group consisting of PT150, PT155, PT156,PT157, PT158, TCY1, combinations thereof, and pharmaceuticallyacceptable salts thereof. The invention provides a method of treatingstress-induced cortisol elevation in a patient in need thereof bydetermining whether the patient has stress-induced cortisol elevationsuitable for GCR (glucocorticoid receptor) antagonist therapy,comprising the steps of a) obtaining a test sample from the patient,optionally at a predetermined time, using a test sample collection unit;b) combining said test sample with a buffering system to form a mixturein a reaction unit; c) measuring a parameter of the mixture to determinea blank measurement; d) combining said test sample and buffer mixturewith a labeled ligand which binds cortisol, wherein the labeled ligandis provided in a label unit, in the reaction unit to produce an assaysolution; or combining said test sample and buffer mixture anddelivering it to a carrier containing a labeled ligand which bindscortisol, wherein the labeled ligand is provided in a label unit, in thereaction unit to produce an assay immobilized complex; e) measuring aparameter of said assay solution or complex; f) comparing themeasurement of the assay solution relative to the blank measurement; g)determining the patient's circulating cortisol levels based on thechange of the measurement; h) comparing the measured cortisol levels toa predetermined reference range cortisol levels, wherein when the levelof cortisol is elevated relative to the predetermined reference range,then the patient has stress-induced cortisol elevation which involveselevated cortisol, and thus has stress-induced cortisol elevationsuitable for GCR antagonist or active agent therapy; and i) when thepatient has stress-induced cortisol elevation suitable for GCRantagonist or active agent therapy, administering at least one GCRantagonist. The invention further provides a method wherein thepatient's test sample is selected from the group consisting of saliva,blood, plasma, serum, urine, other bodily fluids, and combinationsthereof. The invention further provides a method wherein thestress-related cortisol elevation is related to a hospital stay, medicaltreatment, an institutional stay, clinical depression, psychologicalstress, physiological stressors, hypoglycemia, illness, fever, trauma,surgery, fear, pain, physical exertion, or temperature extremes. Theinvention further provides a method wherein the patient is an elderlyindividual. The invention further provides a method wherein the patienthas autism or Asperger's syndrome. The invention further provides amethod wherein the sample is obtained from the patient over more thanone time, and the predetermined time is selected from the groupconsisting of morning, noon, and evening. The invention further providesa method wherein the sample is obtained from the patient overconsecutive days. The invention further provides a method wherein themethod is to determine the circadian cycle of the cortisol levels in thepatient, and the predetermined time is selected from the groupconsisting of hourly, every 4 hours, every 6 hours, every 8 hours, andevery 12 hours. The invention further provides a method wherein thesample is obtained from the patient over consecutive days. The inventionfurther provides a method wherein the predetermined reference range is amedically standard reference range. The invention further provides amethod wherein the predetermined reference range is the patient'spreviously measured level. The invention further provides a methodwherein the ligand is detectably labeled with a moiety selected from thegroup consisting of a radioisotope, a fluorophore, a quencher offluorescence, an enzyme, an affinity tag, and an epitope tag. Theinvention further provides a method wherein said measuring of saidparameter of said mixture and said assay solution is performed using amethod selected from spectroscopic, photochemical, radiochemical,biochemical, enzymatic, immunochemical, chemical label displacement,surface plasmon resonance, fluorescence resonance energy transfer,fluorescence quenching, lateral flow, and fluorescence polarizationmeans. The invention further provides a method wherein the GCRantagonist is selected from the group consisting of PT150, PT155, PT156,PT157, PT158, TCY1, combinations thereof, and pharmaceuticallyacceptable salts thereof.

The invention provides a method of treating post-traumatic stressdisorder in a patient in need thereof by determining whether the patienthas post-traumatic stress disorder suitable for GCR (glucocorticoidreceptor) antagonist therapy, comprising the steps of: a) obtaining atest sample from the patient, optionally at a predetermined time, usinga test sample collection unit; b) combining said test sample with abuffering system to form a mixture in a reaction unit; c) measuring aparameter of the mixture to determine a blank measurement; d) combiningsaid test sample and buffer mixture with a labeled ligand which bindscortisol, wherein the labeled ligand is provided in a label unit, in thereaction unit to produce an assay solution; or combining said testsample and buffer mixture and delivering it to a carrier containing alabeled ligand which binds cortisol, wherein the labeled ligand isprovided in a label unit, in the reaction unit to produce an assayimmobilized complex; e) measuring a parameter of said assay solution orcomplex; f) comparing the measurement of the assay solution relative tothe blank measurement; g) determining the patient's circulating cortisollevels based on the change of the measurement; h) comparing the measuredcortisol levels to a predetermined reference range cortisol levels,wherein when the level of cortisol is elevated relative to thepredetermined reference range, then the patient has post-traumaticstress disorder which involves elevated cortisol, and thus haspost-traumatic stress disorder suitable for GCR (glucocorticoidreceptor) antagonist therapy; and i) when the patient has post-traumaticstress disorder suitable for GCR antagonist or active agent therapy,administering at least one GCR antagonist. The invention furtherprovides a method wherein the patient's test sample is selected from thegroup consisting of saliva, blood, plasma, serum, urine, other bodilyfluids, and combinations thereof. The invention further provides amethod wherein the sample is obtained from the patient over more thanone time, and the predetermined time is selected from the groupconsisting of morning, noon, and evening. The invention further providesa method wherein the sample is obtained from the patient overconsecutive days. The invention further provides a method wherein themethod is to determine the circadian cycle of the cortisol levels in thepatient, and the predetermined time is selected from the groupconsisting of hourly, every 4 hours, every 6 hours, every 8 hours, andevery 12 hours. The invention further provides a method wherein thesample is obtained from the patient over consecutive days. The inventionfurther provides a method wherein the predetermined reference range is amedically standard reference range. The invention further provides amethod wherein the predetermined reference range is the patient'spreviously measured level. The invention further provides a methodwherein the ligand is detectably labeled with a moiety selected from thegroup consisting of a radioisotope, a fluorophore, a quencher offluorescence, an enzyme, an affinity tag, and an epitope tag. Theinvention further provides a method wherein said measuring of saidparameter of said mixture and said assay solution is performed using amethod selected from spectroscopic, photochemical, radiochemical,biochemical, enzymatic, immunochemical, chemical label displacement,surface plasmon resonance, fluorescence resonance energy transfer,fluorescence quenching, lateral flow, and fluorescence polarizationmeans. The invention further provides a method wherein the GCRantagonist is selected from the group consisting of PT150, PT155, PT156,PT157, PT158, TCY1, combinations thereof, and pharmaceuticallyacceptable salts thereof. The invention provides a method of preventionof weight gain in patients using anti-psychotic or anti-depressantmedications in a patient in need thereof, wherein the weight gain issuitable for GCR (glucocorticoid receptor) antagonist therapy,comprising the steps of: a) obtaining a test sample from the patient,optionally at a predetermined time using a test sample collection unit;b) combining said test sample with a buffering system to form a mixturein a reaction unit; c) measuring a parameter of the mixture to determinea blank measurement; d) combining said test sample and buffer mixturewith a labeled ligand which binds cortisol, wherein the labeled ligandis provided in a label unit, in the reaction unit to produce an assaysolution; or combining said test sample and buffer mixture anddelivering it to a carrier containing a labeled ligand which bindscortisol, wherein the labeled ligand is provided in a label unit, in thereaction unit to produce an assay immobilized complex; e) measuring aparameter of said assay solution or complex; f) comparing themeasurement of the assay solution relative to the blank measurement; g)determining the patient's circulating cortisol levels based on thechange of the measurement; h) comparing the measured cortisol levels toa predetermined reference range cortisol levels, wherein when the levelof cortisol is elevated relative to the predetermined reference range,then the patient has weight gain which involves elevated cortisol, whichis suitable for GCR (glucocorticoid receptor) antagonist therapy; and i)when the patient has weight gain suitable for GCR antagonist or activeagent therapy, administering at least one GCR antagonist. The inventionfurther provides a method wherein the patient's test sample is selectedfrom the group consisting of saliva, blood, plasma, serum, urine, otherbodily fluids, and combinations thereof. The invention further providesa method wherein the sample is obtained from the patient over more thanone time, and the predetermined time is selected from the groupconsisting of morning, noon, and evening. The invention further providesa method wherein the sample is obtained from the patient overconsecutive days. The invention further provides a method wherein themethod is to determine the circadian cycle of the cortisol levels in thepatient, and the predetermined time is selected from the groupconsisting of hourly, every 4 hours, every 6 hours, every 8 hours, andevery 12 hours. The invention further provides a method wherein thesample is obtained from the patient over consecutive days. The inventionfurther provides a method wherein the predetermined reference range is amedically standard reference range. The invention further provides amethod wherein the predetermined reference range is the patient'spreviously measured level. The invention further provides a methodwherein the ligand is detectably labeled with a moiety selected from thegroup consisting of a radioisotope, a fluorophore, a quencher offluorescence, an enzyme, an affinity tag, and an epitope tag. Theinvention further provides a method wherein said measuring of saidparameter of said mixture and said assay solution is performed using amethod selected from spectroscopic, photochemical, radiochemical,biochemical, enzymatic, immunochemical, chemical label displacement,surface plasmon resonance, fluorescence resonance energy transfer,fluorescence quenching, lateral flow, and fluorescence polarizationmeans. The invention further provides a method wherein the GCRantagonist is selected from the group consisting of PT150, PT155, PT156,PT157, PT158, TCY1, combinations thereof, and pharmaceuticallyacceptable salts thereof.

The invention provides a method of treating Cushing's syndrome in apatient in need thereof by determining whether the patient has Cushing'ssyndrome suitable for GCR (glucocorticoid receptor) antagonist therapy,comprising the steps of a) obtaining a test sample from the patient,optionally at a predetermined time, using a test sample collection unit;b) combining said test sample with a buffering system to form a mixturein a reaction unit; c) measuring a parameter of the mixture to determinea blank measurement; d) combining said test sample and buffer mixturewith a labeled ligand which binds cortisol, wherein the labeled ligandis provided in a label unit, in the reaction unit to produce an assaysolution; or combining said test sample and buffer mixture anddelivering it to a carrier containing a labeled ligand which bindscortisol, wherein the labeled ligand is provided in a label unit, in thereaction unit to produce an assay immobilized complex; e) measuring aparameter of said assay solution or complex; f) comparing themeasurement of the assay solution relative to the blank measurement; g)determining the patient's circulating cortisol levels based on thechange of the measurement; h) comparing the measured cortisol levels toa predetermined reference range cortisol levels, wherein when the levelof cortisol is elevated relative to the predetermined reference range,then the patient has Cushing's syndrome which involves elevatedcortisol, which is suitable for GCR (glucocorticoid receptor) antagonisttherapy; and i) when the patient has Cushing's syndrome suitable for GCRantagonist or active agent therapy, administering at least one GCRantagonist. The invention further provides a method wherein thepatient's test sample is selected from the group consisting of saliva,blood, plasma, serum, urine, other bodily fluids, and combinationsthereof. The invention further provides a method wherein the sample isobtained from the patient over more than one time, and the predeterminedtime is selected from the group consisting of morning, noon, andevening. The invention further provides a method wherein the sample isobtained from the patient over consecutive days. The invention furtherprovides a method wherein the method is to determine the circadian cycleof the cortisol levels in the patient, and the predetermined time isselected from the group consisting of hourly, every 4 hours, every 6hours, every 8 hours, and every 12 hours. The invention further providesa method wherein the sample is obtained from the patient overconsecutive days. The invention further provides a method wherein thepredetermined reference range is a medically standard reference range.The invention further provides a method wherein the predeterminedreference range is the patients previously measured level. The inventionfurther provides a method wherein the ligand is detectably labeled witha moiety selected from the group consisting of a radioisotope, afluorophore, a quencher of fluorescence, an enzyme, an affinity tag, andan epitope tag. The invention further provides a method wherein saidmeasuring of said parameter of said mixture and said assay solution isperformed using a method selected from spectroscopic, photochemical,radiochemical, biochemical, enzymatic, immunochemical, chemical labeldisplacement, surface plasmon resonance, fluorescence resonance energytransfer, fluorescence quenching, lateral flow, and fluorescencepolarization means. The invention further provides a method wherein theGCR antagonist is selected from the group consisting of PT150, PT155,PT156, PT157, PT158, TCY1, combinations thereof, and pharmaceuticallyacceptable salts thereof.

The invention provides a pharmaceutical composition comprising: (a) GCR(glucocorticoid receptor) antagonist; (b) the pharmaceutical compositionof (a), further comprising at least one pharmaceutically acceptableexcipient; (c) the pharmaceutical composition of (a) or (b), wherein thepharmaceutical composition is formulated or manufactured as a liquid, anelixir, an aerosol, a spray, a powder, a tablet, a pill, a capsule, agel, a geltab, a nanosuspension, a nanoparticle, an extended releasedosage form, or a topical formulation. The invention provides a methodfor treating a condition selected from the group consisting of majordepressive disorder, psychotic depression, stress-induced cortisolelevation, post-traumatic stress disorder, preventing weight gain inpatients using anti-psychotic and anti-depressant medications, or havingCushing's syndrome, in a patient in need of such treatment comprisingadministering the pharmaceutical composition to the patient. Theinvention provides a kit for the treatment, amelioration or preventionof a condition selected from the group consisting of major depressivedisorder, psychotic depression, stress-induced cortisol elevation,post-traumatic stress disorder, preventing weight gain in patients usinganti-psychotic and anti-depressant medications, or having Cushing'ssyndrome, in a patient in need of such treatment comprising: (a) thepharmaceutical composition; and (b) at least one blister package; alidded blister; a blister card or packet; a clamshell; an intravenous(IV) package, N packette or IV container; a tray or a shrink wrapcomprising the pharmaceutical composition of (a) and instructions foruse of the pharmaceutical composition.

The invention provides a product of manufacture comprising a blisterpackage; a lidded blister; a blister card or packet; a clamshell; anintravenous (N) package, IV packette or N container; a tray or a shrinkwrap comprising the pharmaceutical composition and instructions for useof the pharmaceutical composition. The invention further provides amethod wherein the GCR (glucocorticoid receptor) antagonist selectedfrom the group consisting of PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof.

The invention provides a method for treating neoplasia characterized byexpression of a glucocorticoid receptor, in a patient in need of suchtreatment, comprising: administering to said animal or humantherapeutically effective amounts of each of at least oneneoplasia-treating agent and a GCR (glucocorticoid receptor) antagonistselected from the group consisting of PT150, PT155, PT156, PT157, PT158,TCY1, combinations thereof, and pharmaceutically acceptable saltsthereof. The invention further provides a method wherein PT150, PT155,PT156, PT157, PT158, TCY1, combinations thereof, and pharmaceuticallyacceptable salts thereof is administered as an agent which directlyeffects tumor growth, independent of other administered treatmentmodalities, for palliation, remission, or cure. The invention furtherprovides a method wherein said neoplasia treating agent is radiation.The invention further provides a method wherein said neoplasia treatingagent is a biotherapy agent. The invention further provides a methodwherein said neoplasia treating agent is a chemotherapy agent. Theinvention further provides a method wherein said neoplasia treatingagent is a radionuclide. The invention further provides a method whereinthe neoplasia is selected for the group consisting of hepatocellularcarcinoma, esophageal squamous cell carcinoma, breast cancer, pancreaticcancer, squamous cell cancer or adenocarcinoma of the head and neck,colorectal cancer, renal cancer, brain cancer, prostate cancer, smalland non-small cell lung cancer, bladder cancer, bone or joint cancer,uterine cancer, cervical cancer, multiple myeloma, hematopoieticmalignancies, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, skincancer, melanoma, squamous cell carcinoma, leukemia, lung cancer,ovarian cancer, stomach cancer, Kaposi's sarcoma, laryngeal cancer,endocrine carcinomas, cancer of the thyroid gland, cancer of theparathyroid gland, cancer of the pituitary gland, cancer of the adrenalgland, and combinations thereof. The invention further provides a methodwherein the neoplasia expresses multidrug resistance genes when GR isactivated through binding by endogenous cortisol. The invention furtherprovides a method wherein the neoplasia expresses proteins of cellsurvival pathway (including inhibition of apoptosis) genes when GR isactivated through binding by endogenous cortisol. The invention furtherprovides a method wherein the neoplasia expresses genes responsible forepithelial-mesenchymal transition and cell shape maintenance arerepressed when GR is activated through binding by endogenous cortisol.The invention further provides a method wherein the neoplasia expressesgenes involved in signal transduction pathways, lipid/fatty acidmetabolism, inflammation and macrophage regulation, transcriptionalregulation and chromatin remodeling, and cell metabolic pathways. Theinvention further provides a method wherein tumor stem cells (TSC)express GR, blockade of which by PT150, PT155, PT156, PT157, PT158,TCY1, combinations thereof, and pharmaceutically acceptable saltsthereof results in anti-TSC therapy. The invention further provides amethod wherein TSC express multidrug resistance genes when GR isactivated through binding by endogenous cortisol. The invention furtherprovides a method wherein TSC express proteins of cell survival pathways(including inhibition of apoptosis) genes when GR is activated throughbinding by endogenous cortisol. The invention further provides a methodwherein TSC express genes responsible for epithelial-mesenchymaltransition and cell shape maintenance are repressed when GR is activatedthrough binding by endogenous cortisol. The invention further provides amethod wherein TSC express genes involved in signal transductionpathways, lipid/fatty acid metabolism, inflammation and macrophageregulation, transcriptional regulation and chromatin remodeling, andcell metabolic pathways. The invention further provides a method whereinthe neoplasia is chemo-resistant ER/GR+ breast cancer. The inventionfurther provides a method wherein the administration of PT150, PT155,PT156, PT157, PT158, TCY1, combinations thereof, and pharmaceuticallyacceptable salts thereof for GR-blockade, reduces toxicities and sideeffects when given systemically. The invention further provides a methodwherein the PT150, PT155, PT156, PT157, PT158, TCY1, combinationsthereof, and pharmaceutically acceptable salts thereof givensystemically through oral or intravenous routes. The invention furtherprovides a method wherein the PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof istargeted to tumor by intra-arterial infusion to reduce systemic sideeffects of GR blockade. The invention further provides a method whereinthe PT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof is given to accomplish cure orremission of tumor. The invention further provides a method wherein thePT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof is given to accomplishreduction of tumor burden to enhance effectiveness of subsequentsurgical resection. The invention further provides a method wherein thePT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof is given to accomplishreduction of tumor burden to make an unresectable tumor resectable.

The invention provides a method for treatment of neoplasia in a patientcomprising targeted delivery of PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof,wherein the neoplasia expresses multidrug resistance genes when GR isactivated through binding by endogenous cortisol. The invention furtherprovides a method wherein the HCC patient is not a candidate to undergosurgical intervention because tumor is too large or encroaches on liveranatomy in a manner that prevents resection, delivery of PT150, PT155,PT156, PT157, PT158, TCY1, combinations thereof, and pharmaceuticallyacceptable salts thereof prior to ablative or chemotherapy to shrink thetumor and make it resectable. The invention further provides a methodwherein the HCC is present in cirrhosis and the patient is not acandidate for transplantation because of large tumor size,administration of PT150, PT155, PT156, PT157, PT158, TCY1, combinationsthereof, and pharmaceutically acceptable salts thereof making the tumoramenable to ablative or chemotherapy to shrink the tumor and allow thepatient to be eligible for liver transplant. The invention furtherprovides a method wherein the HCC is present in cirrhosis and thepatient is a candidate for transplantation, administration of PT150,PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof making the tumor amenable toablative therapy to manage tumor while patient remains on the livertransplant waitlist.

The invention provides a method for low toxicity chemoprevention bytargeted liver infusion in patients with forms of established cirrhosisthat are high risk for emergence of HCC, including those withpremalignant lesions diagnosed on biopsy or by radiology, comprisingtargeted delivery of PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof tointrahepatic lesions, wherein the targeted delivery of ORG g34517 tointrahepatic lesions prevents emergence of HCC. The invention furtherprovides a method wherein the patient with HCC is not a candidate forundergo surgical intervention. The invention further provides a methodwherein the HCC resides in locations where surgical or ablativeinterventions are not available. The invention further provides a methodwherein the patient with HCC has cirrhosis that is too advanced to makepartial hepatectomy safe. The invention further provides a methodwherein the patient with HCC is too early in their chronic liver diseaseto qualify for transplantation. The invention further provides a methodwherein the HCC is too advanced for localized treatments. The inventionprovides a method for treatment of HCC comprising: a) targeted deliveryof PT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof to intrahepatic lesions,wherein the targeted delivery of PT150, PT155, PT156, PT157, PT158,TCY1, combinations thereof, and pharmaceutically acceptable saltsthereof to intrahepatic lesions improves outcomes of localizedchemo-ablative therapies. The invention further provides a methodwherein the treatment is to help patients qualify for livertransplantation.

The invention provides a method for low toxicity chemoprevention bytargeted liver infusion in patients with forms of established cirrhosisthat are high risk for emergence of HCC, including those withpremalignant lesions diagnosed on biopsy or by radiology comprisingtargeted delivery of PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof tointrahepatic lesions, wherein the targeted delivery of PT150, PT155,PT156, PT157, PT158, TCY1, combinations thereof, and pharmaceuticallyacceptable salts thereof to intrahepatic lesions prevents emergence ofHCC. The invention further provides a method wherein the neoplasia iseSCC.

The invention provides a method for treatment of eSCC in a patient withunresectable eSCC where systemic or targeted administration of PT150,PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof makes tumor responsive toablative or chemotherapies as palliative or curative treatment.

The invention provides method for treatment of eSCC in a patient withunresectable eSCC where systemic or targeted administration of PT150,PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof makes tumor responsive toablative or chemotherapies to shrink the tumor and enhanceresectability. The invention further provides a method wherein theneoplasia-treating agent is a chemotherapeutic agent including but notlimited to gemcitabine, paclitaxel, carboplatin, cisplatin, and5-fluorouracil. The invention further provides a method wherein thetherapeutic effective amount of glucocorticoid administered is about 100to 400 microg/kg body weight per day when administered intravenously.

The invention provides a method for treating neoplasia, in an animal orhuman in need of such treatment, wherein said neoplasia comprisesneoplastic stem cells characterized by expression of a glucocorticoidreceptor, and further characterized by expression of multidrugresistance genes or other stem cell related means of survival when GR isactivated through binding by endogenous cortisol, the method comprising:a) administering to said animal or human a therapeutically effectiveamount of PT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof,and pharmaceutically acceptable salts thereof and b) administering tosaid animal or human a therapeutically effective amount of the at leastone neoplasia-treating agent, wherein said therapeutically effectiveamount of PT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof,and pharmaceutically acceptable salts thereof is an amount sufficient topromote susceptibility of the neoplastic stem cells to at least oneneoplasia-treating agent. The invention further provides a methodwherein said neoplasia-treating agent is radiation selected from thegroup consisting of external beam or radionuclide therapy. The inventionfurther provides a method wherein said neoplasia-treating agent is abiotherapy agent. The invention further provides a method wherein saidneoplasia-treating agent is a chemotherapy agent. The invention furtherprovides a method wherein said neoplasia-treating agent is aradionuclide. The invention further provides a method wherein theneoplasia is selected from the group consisting of hepatocellularcarcinoma, esophageal squamous cell carcinoma, breast cancer, pancreaticcancer, squamous cell cancer or adenocarcinoma of the head and neck,colorectal cancer, renal cancer, brain cancer, prostate cancer, smalland non-small cell lung cancer, bladder cancer, bone or joint cancer,uterine cancer, cervical cancer, multiple myeloma, hematopoieticmalignancies, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, skincancer, melanoma, squamous cell carcinoma, leukemia, lung cancer,ovarian cancer, stomach cancer, Kaposi's sarcoma, laryngeal cancer,endocrine carcinomas, cancer of the thyroid gland, cancer of theparathyroid gland, cancer of the pituitary gland, cancer of the adrenalgland, and combinations thereof. The invention further provides a methodwherein the neoplasia expresses multidrug resistance genes when GR isactivated through binding by endogenous cortisol. The invention providesa pharmaceutical composition for treating neoplasia in a patient whichis characterized by expression of a glucocorticoid receptor, comprising:a) therapeutically effective amounts of at least one neoplasia-treatingagent; b) a GCR (glucocorticoid receptor) antagonist selected from thegroup consisting of PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof; andc) optionally, at least one pharmaceutically acceptable carrier. Theinvention further provides a method wherein said neoplasia-treatingagent is selected from the group consisting of a chemotherapeutic agent,a biotherapeutic agent, a radionuclide agent, and combinations thereof.The invention further provides a method wherein the neoplasia isselected from the group consisting of hepatocellular carcinoma,esophageal squamous cell carcinoma, breast cancer, pancreatic cancer,squamous cell cancer or adenocarcinoma of the head and neck, colorectalcancer, renal cancer, brain cancer, prostate cancer, small and non-smallcell lung cancer, bladder cancer, bone or joint cancer, uterine cancer,cervical cancer, multiple myeloma, hematopoietic malignancies, lymphoma,Hodgkin's disease, non-Hodgkin's lymphoma, skin cancer, melanoma,squamous cell carcinoma, leukemia, lung cancer, ovarian cancer, stomachcancer, Kaposi's sarcoma, laryngeal cancer, endocrine carcinomas, cancerof the thyroid gland, cancer of the parathyroid gland, cancer of thepituitary gland, cancer of the adrenal gland, and combinations thereof.The invention further provides a method, wherein the neoplasia ischemo-resistant ER-GR+ breast cancer. The invention further provides amethod wherein the neoplasia expresses multidrug resistance genes whenGR is activated through binding by endogenous cortisol. The inventionfurther provides a method wherein the chemotherapeutic agent is selectedfrom the group comprising: busulfan, cisplatin, carboplatin,chlorambucil, cyclophosphamide, ifosfamide, dacarbazine (DTIC),mechlorethamine (nitrogen mustard), melphalan carmustine (BCNU)lomustine (CCNU), 5-FU, capecitabine, methotrexate, gemcitabine,cytarabine (ara-C), fludarabine dactinomycin, daunorubicin, doxorubicin(Adriamycin), idarubicin, mitoxantrone, paclitaxel, docetaxel, etoposide(VP-16), vinblastine, vincristine, vinorelbine prednisone,dexamethasone, tamoxifen, fulvestrant, anastrozole, letrozole, megestrolacetate, bicalutamide, flutamide, leuprolide, goserelin, L-asparaginase,and tretinoin, gemcitabine, paclitaxel, carboplatin, 5-FU, andcombinations thereof.

The invention provides a diagnostic kit comprises the followingcomponents: a) test sample collection unit; b) the buffer system unit;c) the reaction unit; and d) the label unit, wherein the components arein a blister package; a lidded blister; a blister card or packet; aclamshell; a tray, or a shrink wrap, and instructions for use of thekit. The invention further provides test sample collection unit whereinthe test sample collection unit comprises a stem integrated with a base,and a head integrated with the stem. The invention further provides atest sample collection unit wherein the stem head comprises a receptorof a sponge-like carrier sufficient to ensure a high void volume toabsorb sufficient saliva, oral fluid or a bodily fluid sample. Theinvention further provides a buffer system unit wherein the buffersystem unit comprises additional components selected from the groupconsisting of viscosity controllers, stabilizers, and combinationsthereof. The invention further provides a reaction unit wherein thereaction unit is adapted to fit in a fluorescent polarization reader.The invention further provides a label unit wherein the label unitcomprises a fluorescence-labeled ligand which binds cortisol, whereinthe fluorescence-labeled ligand which binds cortisol is selected fromthe group consisting of an aptamer, an antibody, an antibody fragment, areceptor, a receptor fragment, a binding polypeptide, a binding peptide,and combinations thereof. The invention further provides a diagnostickit wherein the reader apparatus provides temperature control andon-board mixing as an aid in viscosity control of the reaction to ensurebetter accuracy and precision. The invention further provides adiagnostic kit wherein the reader is a miniaturized, portable apparatusfor measuring fluorescence polarization of a liquid sample by direct orindirect methods. The invention further provides a diagnostic kitwherein system also enables continual monitoring of the patient duringtreatment for assessment of responsiveness to treatment.

The invention provides a method for treating or preventing addiction,addiction induced anxiety, and/or withdrawal symptoms wherein saidmethod comprises administering to a patient in need of such therapy atleast one glucocorticoid receptor antagonist in a therapeuticallyeffective amount. The invention provides a method wherein the at leastone glucocorticoid receptor antagonist and/or active agent is in apharmaceutical preparation. The invention provides a method wherein theglucocorticoid receptor antagonist and/or active agent is selected fromthe group consisting of PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof.

The invention provides a method of treating or preventing addiction,addiction induced anxiety, and/or withdrawal symptoms in a patient inneed thereof, comprising: administering a composition comprising: i) afirst therapeutic agent which is a GCR antagonist, or pharmaceuticallyacceptable salts thereof; ii) at least one or possibly more additionaltherapeutic agent(s) selected from the group consisting of anxiolytics,antidepressants, neuroleptics, or other psychotropic medications andcombinations thereof; and iii) at least one pharmaceutically acceptablecarrier, wherein the first and second therapeutic agents are eachpresent in an amount which, in combination, is a therapeuticallyeffective amount for treating or preventing addiction, addiction inducedanxiety, and/or withdrawal symptoms.

The invention provides a method wherein the second therapeutic agent isselected from the group consisting of at least one anti-anxiety drug, atleast one anti-depressant drug, and at least one neuroleptic medicationand combinations thereof, wherein the at least one anti-anxiety drug isselected from the group consisting of alprazolam, bromazepam, diazepam,lorazepam, clonazepam, temazepam, oxazepam, flunitrazepam, triazolam,chlordiazepoxide, flurazepam, estazolam, nitrazepam, andpharmaceutically acceptable salts, isomers, and mixtures thereof; and/orat least one anti-depressant drug selected from the group consisting ofcitalopram, escitalopram oxalate, fluoxetine, fluvoxamine, paroxetine,sertraline, dapoxetine; venlafaxine and duloxetine; harmaline,iproniazid, isocarboxazid, nialamide, pargyline, phenelzine, selegiline,toloxatone, tranylcypromine, brofaromine, moclobemide; amitriptyline,amoxapine, butriptyline, clomipramine, desipramine, dibenzepin,dothiepin, doxepin, imipramine, iprindole, lofepramine, melitracen,nortriptyline, opipramol, protriptyline, trimipramine; maprotiline,mianserin, nefazodone, trazodone, and pharmaceutically acceptable salts,isomers, and combinations thereof, and/or at least one neuroleptic drugselected from the group consisting of Haloperidol, Droperidol,Benperidol, Triperidol, Melperone, Lenperone, azaperone, Domperidone,risperidone, Chlorpromazine, Fluphenazine, Perphenazine,Prochlorperazine, Thioridazine, Trifluoperazine, Mesoridazine,Periciazine, Promazine, Triflupromazine, Levomepromazine, Promethazine,Pimozide, Cyamemazine, Chlorprothixene, Clopenthixol, Flupenthixol,Thiothixene, Zuclopenthixol, Clozapine, Olanzapine, Risperidone,Quetiapine, Ziprasidone, Amisulpride, Asenapine, Paliperidone,Iloperidone, Zotepine, Sertindole, Lurasidone, Aripiprazole, andpharmaceutically acceptable salts, isomers, and combinations thereof.

The invention provides a method wherein the GCR antagonist is selectedfrom the group consisting of PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof.

The invention provides a method of treating or preventing addiction,addiction induced anxiety, and/or withdrawal symptoms in a patient,comprising the steps of: a) obtaining a test sample from the patient,optionally at a predetermined time, using a test sample collection unit;b) combining said test sample with a buffering system to form a mixturein a reaction unit; c) measuring a parameter of the mixture to determinea blank measurement; d) combining said test sample and buffer mixturewith a labeled ligand which binds cortisol, wherein the labeled ligandis provided in a label unit, in the reaction unit to produce an assaysolution; or combining said test sample and buffer mixture anddelivering it to a carrier containing a labeled ligand which bindscortisol, wherein the labeled ligand is provided in a label unit, in thereaction unit to produce an assay immobilized complex; e) measuring aparameter of said assay solution or complex; f) comparing themeasurement of the assay solution relative to the blank measurement; g)determining the patient's circulating cortisol levels based on thechange of the measurement; h) comparing the measured cortisol levels toa predetermined reference range cortisol levels, wherein when the levelof cortisol is elevated relative to the a predetermined reference range,then the patient has elevated cortisol, and thus is suitable for GCR(glucocorticoid receptor) antagonist therapy; and i) when the patient issuitable for GCR antagonist or active agent therapy, administering atleast one GCR antagonist, thereby treating or preventing addiction,addiction induced anxiety, and/or withdrawal symptoms in the patient.

The invention provides a method wherein the patient's test sample isselected from the group consisting of saliva, blood, plasma, serum,urine, other bodily fluids, and combinations thereof. The inventionprovides a method wherein the sample is obtained from the patient onetime, selected from the group consisting of morning, noon, and evening.The invention provides a method wherein the sample is obtained from thepatient over more than one time, and the predetermined time is selectedto determine the nature of the cortisol circadian rhythm (including itspossible disruption or elimination) from the group consisting ofmorning, noon, and evening. The invention provides a method wherein thesample is obtained from the patient over consecutive days. The inventionprovides a method wherein the method is to determine the circadian cycleof the cortisol levels in the patient, and the predetermined time isselected from the group consisting of hourly, every 4 hours, every 6hours, every 8 hours, and every 12 hours. The invention provides amethod wherein the predetermined reference range is a medically standardreference range. The invention provides a method wherein thepredetermined reference range is the patient's previously measuredlevel. The invention provides a method wherein the ligand is detectablylabeled with a moiety selected from the group consisting of aradioisotope, a fluorophore, a quencher of fluorescence, an enzyme, anaffinity tag, and an epitope tag. The invention provides a methodwherein said measuring of said parameter of said mixture and said assaysolution is performed using a method selected from spectroscopic,photochemical, radiochemical, biochemical, enzymatic, immunochemical,chemical label displacement, surface plasmon resonance, fluorescenceresonance energy transfer, fluorescence quenching, lateral flow, andfluorescence polarization means.

The invention provides a method wherein the GCR (glucocorticoidreceptor) antagonist is selected from the group consisting of PT150,PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof.

The invention provides a kit for treating or preventing addiction,addiction induced anxiety, and/or withdrawal symptoms comprising: (a) apharmaceutical composition comprising at least one glucocorticoidreceptor antagonist and/or active agent in a therapeutically effectiveamount; and (b) at least one blister package; a lidded blister; ablister card or packet; a clamshell; an intravenous (IV) package, IVpackette or IV container; a tray or a shrink wrap comprising thepharmaceutical composition of (a) and instructions for use of thepharmaceutical composition for treating or preventing addiction,addiction induced anxiety, and/or withdrawal symptoms.

The invention provides a product of manufacture for treating orpreventing addiction, addiction induced anxiety, and/or withdrawalsymptoms comprising a blister package; a lidded blister; a blister cardor packet; a clamshell; an intravenous (IV) package, IV packette or IVcontainer; a tray or a shrink wrap comprising a pharmaceuticalcomposition comprising at least one glucocorticoid receptor antagonistand/or active agent, and instructions for treating or preventingaddiction, addiction induced anxiety, and/or withdrawal symptoms.

The invention provides a method for treating bone fracture and/or a bonerelated injury wherein said method comprises administering, to a patientin need of such therapy, at least one glucocorticoid receptor antagonistand/or active agent in a therapeutically effective amount. The inventionprovides a method wherein the at least one glucocorticoid receptorantagonist and/or active agent is in a pharmaceutical preparation. Theinvention provides a method wherein the GCR (glucocorticoid receptor)antagonist is selected from the group consisting of PT150, PT155, PT156,PT157, PT158, TCY1, combinations thereof, and pharmaceuticallyacceptable salts thereof. The invention provides a method wherein theGCR antagonist is administered intravenously, intradermally,intraarterially, intraperitoneally, intralesionally, intracranially,intraarticularly, intraprostaticaly, intrapleurally, intratracheally,intranasally, intrathecally, intravitreally, intravaginally,intrarectally, intratumorally, intramuscularly, intraperitoneally,intraocularly, subcutaneously, subconjunctival, intravesicularlly,mucosally, intrapericardially, intraumbilically, intraocularally,orally, topically, locally, by inhalation, by injection, by infusion, bycontinuous infusion, by localized perfusion, by direct applicationduring surgery, via a catheter, via a lavage, or throughcatheterization, immersion, absorption, or adsorption. The inventionprovides a method wherein the method comprises administering thepharmaceutical composition to tissue surrounding the fracture. Theinvention provides a method wherein administration of the pharmaceuticalcomposition comprises injecting the pharmaceutical composition intotissue surrounding the fracture. The invention provides a method whereinthe glucocorticoid receptor antagonist is selected from the groupconsisting of PT150, PT155, PT156, PT157, PT158, TCY1, combinationsthereof, and pharmaceutically acceptable salts thereof.

The invention provides a kit for treating bone fracture and/or a bonerelated injury comprising: (a) a pharmaceutical composition comprisingat least one glucocorticoid receptor antagonist and/or active agent in atherapeutically effective amount; and (b) at least one blister package;a lidded blister; a blister card or packet; a clamshell; an intravenous(IV) package, IV packette or IV container; a tray or a shrink wrapcomprising the pharmaceutical composition of (a) and instructions foruse of the pharmaceutical composition for treating bone fracture and/ora bone related injury.

The invention provides a product of manufacture for treating bonefracture and/or a bone related injury comprising a blister package; alidded blister; a blister card or packet; a clamshell; an intravenous(IV) package, IV packette or IV container; a tray or a shrink wrapcomprising a pharmaceutical composition comprising at least oneglucocorticoid receptor antagonist and/or active agent, and instructionsfor treating bone fracture and/or a bone related injury.

The invention provides a method for treating or preventing osteoporosis,whether stress induced or related to dysregulated or elevated cortisolof the elderly, wherein said method comprises administering, to apatient in need of such therapy, at least one glucocorticoid receptorantagonist and/or active agent in a therapeutically effective amount.The invention provides a method wherein the at least one glucocorticoidreceptor antagonist and/or active agent is in a pharmaceuticalpreparation.

The invention provides a method wherein the glucocorticoid receptorantagonist and/or active agent is selected from the group consisting ofPT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof. The invention provides amethod wherein the GCR antagonist is administered intravenously,intradermally, intraarterially, intraperitoneally, intralesionally,intracranially, intraarticularly, intranasally, intrarectally,intramuscularly, subcutaneously, orally, topically, locally, byinhalation, by injection, by infusion, by continuous infusion, bylocalized perfusion, by direct application during surgery, via acatheter, via a lavage, or through catheterization, immersion,absorption, or adsorption.

The invention provides a kit for treating or preventing osteoporosis,whether stress induced or related to dysregulated or elevated cortisolof the elderly, comprising: (a) a pharmaceutical composition comprisingat least one glucocorticoid receptor antagonist and/or active agent in atherapeutically effective amount; and (b) at least one blister package;a lidded blister; a blister card or packet; a clamshell; an intravenous(IV) package, IV packette or IV container; a tray or a shrink wrapcomprising the pharmaceutical composition of (a) and instructions foruse of the pharmaceutical composition for treating or preventing stressinduced osteoporosis.

The invention provides a product of manufacture for treating orpreventing osteoporosis, whether stress induced or related todysregulated or elevated cortisol of the elderly, comprising a blisterpackage; a lidded blister; a blister card or packet; a clamshell; anintravenous (IV) package, IV packette or IV container; a tray or ashrink wrap comprising a pharmaceutical composition comprising at leastone glucocorticoid receptor antagonist and/or active agent, andinstructions for treating or preventing osteoporosis, whether stressinduced or related to dysregulated or elevated cortisol of the elderly.

The invention provides a method for enhancing post-transplantfunctioning of stem cells in a patient in need of such treatmentcomprising the steps of: providing stem cells, whether embryonic stemcells, stem cells derived from embryonic stem cells or theirdifferentiated progeny, induced pluripotent stem cells or their progeny,fetal stem cells or their differentiated progeny, and post-natal (adult)stem cells or their differentiated progeny, or of tissues derived fromany of these, treating the stem cells or stem cell-derived,differentiated progeny with at least one GCR antagonist in preparationfor transplantation; and introducing the treated stem cells into thepatient.

The invention provides a method wherein the glucocorticoid receptorantagonist and/or active agent is selected from the group consisting ofPT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof. The invention provides amethod wherein the treated stem cells are administered intravenously,intradermally, intraarterially, intraperitoneally, intralesionally,intracranially, intraarticularly, intraprostaticaly, intrapleurally,intratracheally, intranasally, intrathecally, intravitreally,intravaginally, intrarectally, intratumorally, intramuscularly,intraperitoneally, intraocularly, subcutaneously, subconjunctival,intravesicularly, mucosally, intrapericardially, intraumbilically,intraocularally, orally, topically, locally, by inhalation, byinjection, by infusion, by continuous infusion, by localized perfusion,by direct application during surgery, via a catheter, via a lavage, orthrough catheterization, immersion, absorption, or adsorption. Theinvention provides a pharmaceutical composition for enhancingpost-transplant functioning of stem cells in mammals, comprising: atherapeutically effective amount of the treated stem cells, whetherembryonic stem cells, stem cells derived from embryonic stem cells ortheir differentiated progeny, induced pluripotent stem cells or theirprogeny, fetal stem cells or their differentiated progeny, andpost-natal (adult) stem cells or their differentiated progeny, or oftissues derived from any of these; and a pharmaceutically acceptablemedium or carrier.

The invention provides a method for reducing post-transplant rejectionof a transplanted organ and/or improving graft functioning and survivalcomprising the steps of: providing an organ to be transplanted; treatingthe organ with a GCR antagonist prior to transplantation: and implantingthe organ to a patient in need of such treatment. The invention providesa method wherein the glucocorticoid receptor antagonist is selected fromthe group consisting of PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof. Theinvention provides a method for reducing post-transplantation rejectionand/or improving graft functioning and survival comprising treating apatient in need of such treatment with a GCR antagonist. The inventionprovides a method wherein the glucocorticoid receptor antagonist and/oractive agent is selected from the group consisting of PT150, PT155,PT156, PT157, PT158, TCY1, combinations thereof, and pharmaceuticallyacceptable salts thereof.

The invention provides a method wherein the GCR antagonist isadministered to the organ graft, prior to transplantation,intravascularly, intraarterially, intravenously or in a tissue orimmersion of the whole or part of the organ.

The invention provides a kit for reducing post-transplantation rejectionand/or improving graft functioning and survival comprising: (a) apharmaceutical composition comprising at least one glucocorticoidreceptor antagonist in a therapeutically effective amount; and (b) organpreservation fluid.

The invention provides a kit for reducing post-transplantation rejectionand/or improving graft functioning and survival comprising: (a) apharmaceutical composition comprising at least one glucocorticoidreceptor antagonist in a therapeutically effective amount; and (b) atleast one blister package; a lidded blister; a blister card or packet; aclamshell; an intravenous (IV) package, IV packette or IV container; atray or a shrink wrap comprising the pharmaceutical composition of (a)and instructions for use of the pharmaceutical composition for reducingpost-transplantation rejection.

The invention provides a product of manufacture for reducingpost-transplantation rejection comprising a blister package; a liddedblister; a blister card or packet; a clamshell; an intravenous (IV)package, IV packette or IV container; a tray or a shrink wrap comprisinga pharmaceutical composition comprising at least one glucocorticoidreceptor antagonist, and instructions for reducing post-transplantationrejection and/or improving post-transplant graft functioning andsurvival. The invention provides a method for improving wound healingand preventing excessive scar formation in a patient in need thereof,comprising administering to a mammal a therapeutically effective amountof glucocorticoid receptor antagonist. The invention provides a methodwherein the glucocorticoid receptor antagonist is selected from thegroup consisting of PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof. Theinvention provides a method wherein the wound is treated withglucocorticoid receptor antagonist whereby the glucocorticoid receptorantagonist and/or active agent is administered intravenously,intradermally, intraarterially, intraperitoneally, intralesionally,intracranially, intraarticularly, intraprostaticaly, intrapleurally,intratracheally, intranasally, intrathecally, intravitreally,intravaginally, intrarectally, intratumorally, intramuscularly,intraperitoneally, intraocularly, subcutaneously, subconjunctival,intravesicularlly, mucosally, intrapericardially, intraumbilically,intraocularally, orally, topically, locally, by inhalation, byinjection, by infusion, by continuous infusion, by localized perfusion,by direct application during surgery, via a catheter, via a lavage, orthrough catheterization, immersion, absorption, or adsorption, or bytopical application of bandage, gauze or sutures impregnated withantagonist.

The invention provides a pharmaceutical composition for improving woundhealing and prevention of excessive scarring, comprising: atherapeutically effective amount of the glucocorticoid receptorantagonist, selected from the group consisting of PT150, PT155, PT156,PT157, PT158, TCY1, combinations thereof, and pharmaceuticallyacceptable salts thereof; and a pharmaceutically acceptable medium orcarrier.

The invention provides a pharmaceutical composition for treating orpreventing treatment resistant prostate cancer, comprising: (a)therapeutically effective amounts of at least one androgen receptorantagonist selected from the group consisting of ARN-509, flutamide,nilutamide, enzalutamide, bicalutamide, ketonazole, abiraterone,abiraterone acetate, orteronel, finasteride, dutasteride, bexlosteride,izonsteride, turosteride, episteride, dexamethasone, prednisone,leuprolide, goserelin, triptorelin, histrelin, estrogen, MDV3100,Cyproterone acetate, Spironolactone, flutamide, hydroxyflutamide, andcombinations thereof; (b) therapeutically effective amounts of at leastone GCR (glucocorticoid receptor) antagonist selected from the groupconsisting of PT150, PT155, PT156, PT157, PT158, TCY1, combinationsthereof, and pharmaceutically acceptable salts thereof, and combinationsthereof; and (c) optionally, at least one pharmaceutically acceptablecarrier. The invention provides a kit for treating or preventingtreatment resistant prostate cancer comprising: (a) therapeuticallyeffective amounts of at least one GCR (glucocorticoid receptor)antagonist selected from the group consisting of PT150, PT155, PT156,PT157, PT158, TCY1, combinations thereof, and pharmaceuticallyacceptable salts thereof, and combinations thereof; (b) therapeuticallyeffective amounts of at least one androgen receptor antagonist selectedfrom the group consisting of ARN-509, flutamide, nilutamide,enzalutamide, bicalutamide, ketonazole, abiraterone, abirateroneacetate, orteronel, finasteride, dutasteride, bexlosteride, izonsteride,turosteride, episteride, dexamethasone, prednisone, leuprolide,goserelin, triptorelin, histrelin, estrogen, MDV3100, Cyproteroneacetate, Spironolactone, flutamide, hydroxyflutamide, and combinationsthereof; (c) optionally, at least one pharmaceutically acceptablecarrier; and (d) at least one blister package; a lidded blister; ablister card or packet; a clamshell; an intravenous (IV) package, IVpackette or IV container; a tray or a shrink wrap comprising thepharmaceutical composition of (a) and instructions for use of thepharmaceutical composition for treating or preventing treatmentresistant prostate cancer.

The invention provides a product of manufacture for treating orpreventing treatment resistant prostate cancer comprising a blisterpackage; a lidded blister; a blister card or packet; a clamshell; anintravenous (IV) package, IV packette or IV container; a tray or ashrink wrap comprising the pharmaceutical composition of claim 1, andinstructions for use of the pharmaceutical composition for treating orpreventing treatment resistant prostate cancer.

The invention provides a method for treating or preventing treatmentresistant prostate cancer, in a patient in need of such treatment orprevention, comprising: administering to said patient therapeuticallyeffective amounts of each of: (a) therapeutically effective amounts ofat least one androgen receptor antagonist selected from the groupconsisting of ARN-509, flutamide, nilutamide, enzalutamide,bicalutamide, ketonazole, abiraterone, abiraterone acetate, orteronel,finasteride, dutasteride, bexlosteride, izonsteride, turosteride,episteride, dexamethasone, prednisone, leuprolide, goserelin,triptorelin, histrelin, estrogen, MDV3100, Cyproterone acetate,Spironolactone, flutamide, hydroxyflutamide, and combinations thereof;and (b) therapeutically effective amounts of at least one GCR(glucocorticoid receptor) antagonist selected from the group consistingof PT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof, and combinations thereof. Theinvention provides a method wherein the GCR (glucocorticoid receptor)antagonist is PT150, PT155, PT156, PT157, PT158, TCY1, combinationsthereof, and pharmaceutically acceptable salts thereof. The inventionprovides a method wherein PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof isadministered as an agent which directly effects tumor growth,independent of other administered treatment modalities, for palliation,remission, or cure. The invention provides a method wherein the PT150,PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof given systemically throughoral or intravenous routes. The invention provides a method wherein thePT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof is targeted to tumor byintra-arterial infusion to reduce systemic side effects of GR blockade.The invention provides a method wherein the PT150, PT155, PT156, PT157,PT158, TCY1, combinations thereof, and pharmaceutically acceptable saltsthereof is given to accomplish cure or remission of tumor. The inventionprovides a method wherein the PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof isgiven to accomplish reduction of tumor burden to enhance effectivenessof subsequent surgical resection. The invention provides a methodwherein the PT150, PT155, PT156, PT157, PT158, TCY1, combinationsthereof, and pharmaceutically acceptable salts thereof is given toaccomplish reduction of tumor burden to make an unresectable tumorresectable.

The invention provides a pharmaceutical composition for treatingneoplasia in a patient in need thereof, comprising: (a) therapeuticallyeffective amounts of at least one PARP inhibitor selected from the groupconsisting of4-[[3-[4-(cyclopropanecarbonyl)piperazine-1-carbonyl]-4-fluorophenylFmet-hyl]-2H-phthalazin-1-one(Compound B, i.e., Olaparib), 4-iodo-3-nitrobenzamide (Iniparib),2-[(2R)-2-methylpyrrolidin-2-yl]-1H-benzimidazole-4-carboxamide(ABT-888),8-Fluoro-2-{4-[(methylamino)methyl]-phenyl}-1,3,4,5-tetrahydro-6H-azepino-[5,4,3-cd]indol-6-one(AG014699), 4-methoxy-carbazole (CEP 9722),2-[4-[(3S)-piperidin-3-yl]phenyl]indazole-7-carboxamide hydrochloride(MK 4827), and 3-aminobenzamide, Iniparib, Olaparib, Rucaparib,Veliparib, CEP-9722, MK4827, BMN-673, pharmaceutically acceptable saltsthereof, and combinations thereof; (b) therapeutically effective amountsof at least one GCR (glucocorticoid receptor) antagonist selected fromthe group consisting of PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof; and(c) optionally, at least one pharmaceutically acceptable carrier.

The invention provides a kit for treating or preventing neoplasiacomprising: (a) therapeutically effective amounts of at least one PARPinhibitor selected from the group consisting of4-[[3-[4-(cyclopropanecarbonyl)piperazine-1-carbonyl]-4-fluorophenyl]-met-hyl]-2H-phthalazin-1-one(Compound B, i.e., Olaparib), 4-iodo-3-nitrobenzamide (Iniparib),2-[(2R)-2-methylpyrrolidin-2-yl]-1H-benzimidazole-4-carboxamide(ABT-888),8-Fluoro-2-{4-[(methylamino)methyl]-phenyl}-1,3,4,5-tetrahydro-6H-azepino-[5,4,3-cd]indol-6-one (AG014699), 4-methoxy-carbazole (CEP 9722),2-[4-[(3S)-piperidin-3-yl]phenyl]indazole-7-carboxamide hydrochloride(MK 4827), and 3-aminobenzamide, Iniparib, Olaparib, Rucaparib,Veliparib, CEP-9722, MK4827, BMN-673, pharmaceutically acceptable saltsthereof, and combinations thereof; and (b) therapeutically effectiveamounts of at least one GCR (glucocorticoid receptor) antagonistselected from the group consisting of PT150, PT155, PT156, PT157, PT158,TCY1, combinations thereof, and pharmaceutically acceptable saltsthereof; and (c) optionally, at least one pharmaceutically acceptablecarrier; and (d) at least one blister package; a lidded blister; ablister card or packet; a clamshell; an intravenous (IV) package, IVpackette or IV container; a tray or a shrink wrap comprising thepharmaceutical composition of (a) and instructions for use of thepharmaceutical composition for treating or preventing neoplasia. Theinvention provides a product of manufacture for treating or preventingneoplasia comprising a blister package; a lidded blister; a blister cardor packet; a clamshell; an intravenous (IV) package, IV packette or IVcontainer; a tray or a shrink wrap comprising the pharmaceuticalcomposition of claim 12, and instructions for use of the pharmaceuticalcomposition for treating or preventing neoplasia.

The invention provides a method for treating or preventing neoplasia ina patient in need of such treatment or prevention, comprising:administering to said patient: (a) therapeutically effective amounts ofat least one PARP inhibitor selected from the group consisting of4-[[3-[4-(cyclopropanecarbonyl)piperazine-1-carbonyl]-4-fluorophenyl]-met-hyl]-2H-phthalazin-1-one(Compound B, i.e., Olaparib), 4-iodo-3-nitrobenzamide (Iniparib),2-[(2R)-2-methylpyrrolidin-2-yl]-1H-benzimidazole-4-carboxamide(ABT-888),8-Fluoro-2-{4-[(methylamino)methyl]-phenyl}-1,3,4,5-tetrahydro-6H-azepino-[5,4,3-cd]indol-6-one (AG014699), 4-methoxy-carbazole (CEP 9722),2-[4-[(3S)-piperidin-3-yl]phenyl]indazole-7-carboxamide hydrochloride(MK 4827), and 3-aminobenzamide, Iniparib, Olaparib, Rucaparib,Veliparib, CEP-9722, MK4827, BMN-673, pharmaceutically acceptable saltsthereof, and combinations thereof; (b) therapeutically effective amountsof at least one GCR (glucocorticoid receptor) antagonist or active agentselected from the group consisting of PT150, PT155, PT156, PT157, PT158,TCY1, combinations thereof, and pharmaceutically acceptable saltsthereof; and (c) optionally, at least one pharmaceutically acceptablecarrier. The invention provides a method wherein the GCR (glucocorticoidreceptor) antagonist is PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof. Theinvention provides a method wherein PT150, PT155, PT156, PT157, PT158,TCY1, combinations thereof, and pharmaceutically acceptable saltsthereof is administered as an agent which directly effects tumor growth,independent of other administered treatment modalities, for palliation,remission, or cure. The invention provides a method wherein the PT150,PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof given systemically throughoral or intravenous routes. The invention provides a method wherein theneoplasia is selected for the group consisting of Adenocarcinomas of thehead and neck (including salivary glands and oral cavity),gastrointestinal tract (including pharynx, esophagus, stomach, smallintestine, large intestine, anus), lung, liver (including hepatocellularcarcinoma, cholangiocarcinoma, and mixed tumors), extrahepatic biliarytract and gallbladder, pancreas (including ductal and acinar types),genitourinary tracts (ovaries, fallopian tubes, endometrium, cervix, andvagina, ureters, urinary bladder, testicles, epididymis, prostate), andskin adnexa; squamous cell carcinomas of the head and neck (includingsalivary glands and oral cavity), gastrointestinal tract (includingpharynx, esophagus, anus), lung, intrahepatic and extrahepatic biliarytree (including gallbladder), pancreas, genitourinary tracts (includingendometrium, cervix, vagina, ureters, urinary bladder, testicles,epididymis, prostate), and skin adnexa; germ cell tumors (includingmalignant teratoma, embryonal carcinoma, struma ovarii, yolk sac tumor,seminoma, choriocarcinoma); sarcomas (including leiomyosarcomas,rhabdomyosarcomas, angiosarcomas, hemangioendotheliomas, liposarcomas,chondosarcomas, fibrosarcomas, Ewing sarcoma, malignant nerve sheathetumors, alveolar soft part sarcomas, clear cell sarcomas, synovialsarcoma, osteosarcomas); malignancies of the central nervous system(including astrocytomas, oligodendroglioma, glioblastoma,medulloblastoma); salivary gland malignancies (including adenoid cysticcarcinoma, adenosquamous carcinoma, clear cell carcinoma,cystadenocarcinoma, mucoepidermoid carcinoma); mixed type carcinomas(including hepatocellular-cholangiocarcinomas, carcinosarcomas, mixedadenoneurondocrine carcinomas, adenosquamous carcinomas); hepatocellularcarcinoma; blastic malignancies (including hepatoblastoma,neuroblastoma, ganglioneuroblastoma, nephroblastoma); renal cellcarcinomas; neuroendocrine carcinomas; thyroid carcinomas (includingpapillary, follicular, medullary, anaplastic carcinomas); parathyroidgland carcinomas, pituitary gland carcinomas, adrenal gland carcinomas(including adrenocortical carcinomas, pheochromocytoma), andcombinations thereof. The invention provides a method wherein the PT150,PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof is targeted to tumor byintra-arterial infusion to reduce systemic side effects of GR blockade.The invention provides a method wherein the PT150, PT155, PT156, PT157,PT158, TCY1, combinations thereof, and pharmaceutically acceptable saltsthereof is given to accomplish cure or remission of tumor. The inventionprovides a method wherein the PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof isgiven to accomplish reduction of tumor burden to enhance effectivenessof subsequent surgical resection. The invention provides a methodwherein the PT150, PT155, PT156, PT157, PT158, TCY1, combinationsthereof, and pharmaceutically acceptable salts thereof is given toaccomplish reduction of tumor burden to make an unresectable tumorresectable.

The invention provides a pharmaceutical composition for treating orpreventing infection related to acute or chronic injury or disease in apatient in need thereof, comprising: (a) therapeutically effectiveamounts of at least one GCR (glucocorticoid receptor) antagonistselected from the group consisting of PT150, PT155, PT156, PT157, PT158,TCY1, combinations thereof, and pharmaceutically acceptable saltsthereof and (b) optionally, at least one pharmaceutically acceptablecarrier.

The invention provides a method for treating or preventing infectionrelated to acute or chronic injury or disease in a patient in need ofsuch treatment, comprising: administering to said animal or humantherapeutically effective amounts of each of: (a) therapeuticallyeffective amounts of at least one GCR (glucocorticoid receptor)antagonist selected from the group consisting of PT150, PT155, PT156,PT157, PT158, TCY1, combinations thereof, and pharmaceuticallyacceptable salts thereof; (b) optionally, at least one pharmaceuticallyacceptable carrier.

The invention provides a method for treating or preventing infectionrelated to acute or chronic injury or disease wherein said methodcomprises administering to a patient in need of such therapy at leastone glucocorticoid receptor antagonist in a therapeutically effectiveamount.

The invention provides a method wherein the at least one glucocorticoidreceptor antagonist and/or active agent is in a pharmaceuticalpreparation.

The invention provides a method wherein the glucocorticoid receptorantagonist and/or active agent is selected from the group consisting ofPT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof.

The invention provides a method wherein the acute or chronic injury ordisease is selected from the group consisting of vascular events,stroke, cardiac arrest, acute limb infarction accident/battle fieldtrauma, traumatic limb, hip, brain injuries, post-surgical trauma, majororthopedic, thoracic, abdominal, neurological surgeries.

The invention provides a kit for treating or preventing infectionrelated to acute or chronic injury or disease comprising at least oneblister package; a lidded blister; a blister card or packet; aclamshell; an intravenous (IV) package, IV packette or IV container; atray or a shrink wrap comprising the pharmaceutical composition of claim24, and instructions for use of the pharmaceutical composition fortreating or preventing infection related to acute or chronic injury ordisease.

The invention provides a product of manufacture for treating orpreventing infection related to acute or chronic injury or diseasecomprising a blister package; a lidded blister; a blister card orpacket; a clamshell; an intravenous (IV) package, IV packette or IVcontainer; a tray or a shrink wrap comprising a pharmaceuticalcomposition comprising the pharmaceutical composition of claim 24, andinstructions for use of the pharmaceutical composition for treating orpreventing infection related to acute or chronic injury or disease.

The invention provides a pharmaceutical composition for treating orpreventing impaired short term memory performance in a patient in needthereof, comprising: (a) therapeutically effective amounts of at leastone GCR (glucocorticoid receptor) antagonist selected from the groupconsisting of PT150, PT155, PT156, PT157, PT158, TCY1, combinationsthereof, and pharmaceutically acceptable salts thereof; and (b)optionally, at least one pharmaceutically acceptable carrier.

The invention provides a method for treating or preventing impairedshort term memory performance in a patient in need thereof, comprising:administering to said animal or human therapeutically effective amountsof each of: (a) therapeutically effective amounts of at least one GCR(glucocorticoid receptor) antagonist selected from the group consistingof PT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof; (b) optionally, at least onepharmaceutically acceptable carrier.

The invention provides a kit for treating or preventing infectionrelated to acute or chronic injury or disease comprising at least oneblister package; a lidded blister; a blister card or packet; aclamshell; an intravenous (IV) package, IV packette or IV container; atray or a shrink wrap comprising the pharmaceutical composition of claim32, and instructions for use of the pharmaceutical composition fortreating or preventing infection related to acute or chronic injury ordisease.

The invention provides a product of manufacture for treating orpreventing infection related to acute or chronic injury or diseasecomprising a blister package; a lidded blister; a blister card orpacket; a clamshell; an intravenous (IV) package, IV packette or IVcontainer; a tray or a shrink wrap comprising a pharmaceuticalcomposition comprising the pharmaceutical composition of claim 32, andinstructions for use of the pharmaceutical composition for treating orpreventing infection related to acute or chronic injury or disease.

The invention provides for the use of the compositions of the inventionfor the production of a medicament for treating the indications as setforth herein.

In accordance with a further embodiment, the present invention providesa use of the pharmaceutical compositions described above, an amounteffective for use in a medicament, and most preferably for use as amedicament for treating a disease or disorder in a subject.

In accordance with yet another embodiment, the present inventionprovides a use of the pharmaceutical compositions described above, andat least one additional therapeutic agent, in an amount effective foruse in a medicament, and most preferably for use as a medicament fortreating a disease or disorder associated with disease in a subject.

The invention provides a method for treating or preventing infectionafter ischemic stroke wherein said method comprises administering to apatient in need of such therapy at least one glucocorticoid receptorantagonist in a therapeutically effective amount.

The invention provides a method wherein the at least one glucocorticoidreceptor antagonist and/or active agent is in a pharmaceuticalpreparation.

The invention provides a method wherein the glucocorticoid receptorantagonist and/or active agent is selected from the group consisting ofPT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof.

The invention provides a method of treating or preventing infectionsecondary to CNS injury in a patient in need thereof, comprisingadministering at least one GCR antagonist in a therapeutically effectiveamount.

The invention provides a method wherein the CNS injury is selected fromthe group consisting of stroke, neuronal damage resulting from headtrauma, epilepsy, pain, migraine, a mood disorder, schizophrenia, aneurodegenerative disorder, depression, anxiety, a psychosis,hypertension or cardiac arrhythmia, and combinations thereof.

The invention provides a kit for treating or preventing infection afterischemic stroke comprising: (a) a pharmaceutical composition comprisingat least one glucocorticoid receptor antagonist in a therapeuticallyeffective amount; and (b) at least one blister package; a liddedblister; a blister card or packet; a clamshell; an intravenous (IV)package, IV packette or IV container; a tray or a shrink wrap comprisingthe pharmaceutical composition of (a) and instructions for use of thepharmaceutical composition for treating or preventing infection afterischemic stroke.

The invention provides a product of manufacture for treating orpreventing infection after ischemic stroke comprising a blister package;a lidded blister; a blister card or packet; a clamshell; an intravenous(IV) package, IV packette or IV container; a tray or a shrink wrapcomprising a pharmaceutical composition comprising at least oneglucocorticoid receptor antagonist, and instructions for use of thepharmaceutical composition for treating or preventing infection afterischemic stroke.

The invention provides a method for reducing infarct severity andimproving long term outcome after ischemic stroke wherein said methodcomprises administering, to a patient in need of such therapy, at leastone glucocorticoid receptor antagonist in a therapeutically effectiveamount.

The invention provides a method wherein the at least one glucocorticoidreceptor antagonist and/or active agent is in a pharmaceuticalpreparation.

The invention provides a method wherein the glucocorticoid receptorantagonist and/or active agent is selected from the group consisting ofPT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof.

The invention provides a method of treating or preventing infectionsecondary to CNS injury in a patient in need thereof, comprisingadministering at least one GCR antagonist in a therapeutically effectiveamount.

The invention provides a method for improving long term outcomesecondary to CNS injury in a patient in need thereof, comprisingadministering at least one GCR antagonist.

The invention provides a method wherein the CNS injury is selected fromthe group consisting of stroke, neuronal damage resulting from headtrauma, epilepsy, pain, migraine, a mood disorder, schizophrenia, aneurodegenerative disorder, depression, anxiety, a psychosis,hypertension or cardiac arrhythmia, and combinations thereof.

The invention provides a kit for reducing infarct severity and improvinglong term outcome after ischemic stroke comprising: (a) a pharmaceuticalcomposition comprising at least one GCR antagonist; and (b) at least oneblister package; a lidded blister; a blister card or packet; aclamshell; an intravenous (IV) package, IV packette or IV container; atray or a shrink wrap comprising the pharmaceutical composition of (a)and instructions for use of the pharmaceutical composition for treatingor preventing infection after ischemic stroke.

The invention provides a product of manufacture for reducing infarctseverity and improving long term outcome after ischemic strokecomprising a blister package; a lidded blister; a blister card orpacket; a clamshell; an intravenous (IV) package, IV packette or IVcontainer; a tray or a shrink wrap comprising a pharmaceuticalcomposition comprising at least one glucocorticoid receptor antagonist,and instructions for use of the pharmaceutical composition for treatingor preventing infection after ischemic stroke.

The invention provides a glucocorticoid receptor antagonist selectedfrom the group consisting of PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof foruse in the preparation of a medicament for use in treating or preventinga disease or condition as set forth herein in a patient. The inventionprovides a pharmaceutical composition comprising a glucocorticoidreceptor antagonist selected from the group consisting of PT150, PT155,PT156, PT157, PT158, TCY1, combinations thereof, and pharmaceuticallyacceptable salts thereof, for use in the preparation of a medicament foruse in topical application to treat and/or prevent a disease orcondition as set forth herein in a patient. The invention provides amethod for treating and/or preventing a disease or condition as setforth herein in a patient, wherein said method comprises: selecting apatient in need of treating and/or preventing said disease or conditionas set forth herein; administering to the patient a composition of theinvention in a therapeutically effective amount, thereby treating and/orpreventing said disease in said patient.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

The invention will be described in conjunction with the followingdrawings in which like reference numerals designate like elements andwherein:

FIG. 1 shows Bio-activation by human flavin-dependent monooxygenases(hFMOs) and GRE DNA-trapping of PT155. The thiosemicarbazone moiety inPT155 can be oxidized by hFMOs (hFMO1, hFMO2.1, hFMO3) with molecularoxygen over the sulfenic acid to the PT155 carbodiimide metabolite whichcould be co-transported into the host cell nucleus by hGRalpha:hGRalphasubunit 1-(PT155 carbodiimide metabolite)=hGRalphasubunit2-PT150. The carbodiimide moiety adds to one amino group of proviral GREDNA yielding an N-amidinohydrazone (amidrazone)-group, thereby inducingproviral DNA damage.

FIG. 2 shows Covalent Inactivation of HIV-1 host DNA-integrated proviralGRE DNA.

FIG. 3 is a chart showing Inhibition of HIV-1 LAV Replication in OM-10.1by TMZ.

FIG. 4 is a chart showing Inhibition of HIV-1 LAV Replication in OM-10.1by PT155.

FIG. 5 shows Flavivirus (+)-ssRNA genome-encoded polyprotein with thelocations of 5′-noncoding region (5′-NCR) and 3′-noncoding region(3′-NCR at 3′-OH). This graphic shows a typical Flaviviruspositive-sense single-stranded RNA [(+)-ssRNA] (length about 11,000nucleotides) genome encoding a polyprotein. The (+)-ssRNA genome isshown with short 5′-capped (left) 5′-noncoding region (5′-NCR), and the3′-noncoding region [3′-NCR; in green at the right end (3′-OH) of thegenome]. The host cell and viral protease cleavage sites in thepolyprotein leading to mature flaviviral proteins are indicated (hostcell signal peptidase; host cell Golgi protease; viral NS3 protease).

FIG. 6 shows Zika virus (+)-ssRNA genome-encoded polyprotein with thelocations of 3′-noncoding region (3′-NCR at 3′-OH) and the origin ofsfRNA. This graphic shows Zika virus (isolate Zikavirus/M.mulatta-tc/UGA/1947/MR-766) positive-sense single-stranded RNA[(+)-ssRNA] (10,795 nt) genome (GenBank: KU955594.1) encoding apolyprotein. The (+)-ssRNA genome is shown with the 3′-noncoding region[3′-NCR; in green at the right end (3′-OH) of the genome]. The origin ofthe subgenomic flavivirus RNA (sfRNA) by human host cell XRN1 5′ 3′-2525exoribonuclease is indicated.

FIG. 7 is a Schematic representation of the 3′-NCR of West Nile virus.FIG. 7(A) Schematic representation of the 3′-NCR of West Nile virusdemonstrating the arrangement of stem-loops (SL) and pseudoknots (PK)and the sfRNA 5′-terminus. FIG. 7(B) Schematic representationdemonstrating the predicted conservation of the SL-II/PK1-like RNAstructure within the 3′-NCR of divergent members of the genus Flavivirus

FIG. 8 shows carbodiimide metabolite can react with amino acid residuesin target proteins of, for example, Mycobacterium tuberculosis (refs 11,14), or with human hepatic/extrahepatic glutathione R—SH thiol group formetabolic detoxification.

FIG. 9 shows thiosemicarbazones like thiacetazone(p-acetamidobenzaldehyde thiosemicarbazone), a cheap, second-lineantitubercular substance discovered by Nobel laureate Gerhard Domagk in1946 (ref. 13), are bio-activated by human flavin-containingmonooxygenases (hFMO1, hFMO2.1, hFMO3) into a sulfenic acid (R—S—OH), asulfinic acid [R—(S═O)—OH], and a carbodiimide derivative (R—N═C═N—H).

FIG. 10 shows the 700.35 MHz proton (1H) nuclear magnetic resonance(NMR) spectrum of PT155 dissolved in DMSO-d6.

FIG. 11 shows the 700.35 MHz proton (1H) nuclear magnetic resonance(NMR) spectrum of PT156 dissolved in DMSO-d6.

FIG. 12 shows the 700.35 MHz proton (1H) nuclear magnetic resonance(NMR) spectrum of highly purified ORG34517 (PT150) dissolved in DMSO-d6.

FIG. 13 shows the 700.35 MHz proton (1H) nuclear magnetic resonance(NMR) spectrum of PT157 dissolved in DMSO-d6.

FIG. 14 shows the 700.35 MHz proton (1H) nuclear magnetic resonance(NMR) spectrum of PT158 dissolved in DMSO-d6.

FIG. 15 shows enantiomers of ORG34517 (PT150).

FIG. 16 shows Zika virus NS2A protein contains the LXXLL glucocorticoidreceptor coactivator sequence. A ternary complex Mov34-NS2A_(Zika)-hGRαcan be formed in case of Zika virus infection, which in turn isessential to Zika virus RNA replication. Zika virus 3′-NCR requires theMov34-NS2A_(Zika)-hGRα axis to replicate optimally in human cells, thatmeans forming a functional replicase complex at the 3′-NCR fornegative-strand (−)-ssRNA synthesis. This also requires thephysiological human glucocorticosteroid cortisol (hydrocortisone) boundto hGRα in this axis. Glucocorticoid antagonists, especially PT155,prevent activation of Zika virus 3′-NCR required for negative-strand(−)-ssRNA synthesis.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to the use of, for example, a glucocorticoidreceptor antagonist, optionally in combination with another agent, fortreating or preventing treatment resistant prostate cancer, treating orpreventing neoplasia, and/or treating or preventing infection related toacute or chronic injury or disease.

As used herein, the term “effective amount” refers to the amount of atherapy that is sufficient to result in the prevention of thedevelopment, recurrence, or onset of a disease or condition, such asneoplasia or infection, and one or more symptoms thereof, to enhance orimprove the prophylactic effect(s) of another therapy, reduce theseverity, the duration of a disease or condition, such as neoplasia orinfection, ameliorate one or more symptoms of a disease or conditionsuch as neoplasia or infection, prevent the advancement of a disease orcondition, such as neoplasia or infection, cause regression of a diseaseor condition, such as neoplasia or infection, and/or enhance or improvethe therapeutic effect(s) of another therapy. An amount is “effective”as used herein, when the amount provides an effect in the subject. Asused herein, the term “effective amount” means an amount of a compoundor composition sufficient to significantly induce a positive benefit,including independently or in combinations the benefits disclosedherein, but low enough to avoid serious side effects, i.e., to provide areasonable benefit to risk ratio, within the scope of sound judgment ofthe skilled artisan. For those skilled in the art, the effective amount,as well as dosage and frequency of administration, may easily bedetermined according to their knowledge and standard methodology ofmerely routine experimentation based on the present disclosure.

As used herein, the phrase “pharmaceutically acceptable” means approvedby a regulatory agency of the federal or a state government, or listedin the U.S. Pharmacopeia, European Pharmacopeia, or other generallyrecognized pharmacopeia for use in animals, and more particularly, inhumans.

As used herein, the terms “prevent,” “preventing” and “prevention” inthe context of the administration of a therapy to a subject refer to theprevention or inhibition of the recurrence, onset, and/or development ofa disease or condition, such as neoplasia, viral infection, latent viralinfections, or a symptom thereof in a subject resulting from theadministration of a therapy (e.g., a prophylactic or therapeutic agent),or a combination of therapies (e.g., a combination of prophylactic ortherapeutic agents).

As used herein, the terms “subject” and “patient” are usedinterchangeably. As used herein, the term “patient” refers to an animal,preferably a mammal such as a non-primate (e.g., cows, pigs, horses,cats, dogs, rats etc.) and a primate (e.g., monkey and human), and mostpreferably a human. In some embodiments, the subject is a non-humananimal such as a farm animal (e.g., a horse, pig, or cow) or a pet(e.g., a dog or cat). In a specific embodiment, the subject is anelderly human. In another embodiment, the subject is a human adult. Inanother embodiment, the subject is a human child. In yet anotherembodiment, the subject is a human infant.

As used herein, the terms “therapies” and “therapy” can refer to anymethod(s), composition(s), and/or agent(s) that can be used in theprevention, treatment and/or management of a disease or condition, suchas neoplasia or viral infection, or one or more symptoms thereof. Asused herein, the terms “treat,” “treatment,” and “treating” in thecontext of the administration of a therapy to a subject refer to thereduction or inhibition of the progression and/or duration of a diseaseor condition, such as neoplasia or viral infection, the reduction oramelioration of the severity of a disease or condition, such asneoplasia or infection, and/or the amelioration of one or more symptomsthereof resulting from the administration of one or more therapies.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, concentration or percentage ismeant to encompass variations of in some embodiments ±20%, in someembodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, insome embodiments ±0.5%, and in some embodiments ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethod.

As used herein, ranges can be expressed as from “about” one particularvalue, and/or to “about” another particular value. It is also understoodthat there are a number of values disclosed herein, and that each valueis also herein disclosed as “about” that particular value in addition tothe value itself. For example, if the value “10” is disclosed, then“about 10” is also disclosed. It is also understood that each unitbetween two particular units are also disclosed. For example, if 10 and15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

The term “androgenergic antagonist” refers to agents that can preventandrogens from expressing their biological effects on responsivetissues. These agents alter the androgen pathway by blocking theappropriate receptors, competing for binding sites on the cell'ssurface, or affecting androgen production. Androgenergic antagonist canbe prescribed to treat an array of diseases and disorders. In men, theseagents are most frequently used to treat prostate cancer. In women,these agents are used to decrease levels of male hormones causingsymptoms of hyperandrogenism. Androgenergic antagonist present in theenvironment have become a topic of concern. Many industrial chemicals,pesticides and insecticides exhibit antiandrogenic effects. Non-limitingexamples of the androgenergic antagonist include, but not limited to,allylestrenol, oxendolone, osaterone acetate, bicalutamide, steroidal,anti-androgergic agents, medroxyprogesterone (MPA), cyproterone,cyproterone acetate (CPA), dienogest, flutamide, nilutamide,spironolactone, Salpha-reductase inhibitors, dutasteride, finasteride,salts thereof, gold nanoparticles thereof, combinations thereof, and thelike. In some embodiments of the present invention, examples of theandrogenergic antagonist includes, but not limited to a goldnanoparticle of alpha-bicalutamide, or a gold nanoparticle ofbeta-bicalutamide.

The present invention relates to the use of GR antagonists (e.g., ORG34517, PT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof,and pharmaceutically acceptable salts) for the prevention or addictioninduced anxiety and withdrawal side effects as a therapeutic, for woundhealing and transplants, for the prevention or treatment of stressinduced osteoporosis and for the rapid healing of bone related injuries,and regenerative therapy.

This invention relates to a low cost rapid response diagnostic system todetermine salivary cortisol levels in patients selected as potentialcandidates for GCR (glucocorticoid receptor) antagonist therapyutilizing a GCR antagonist, such as ORG 34517, PT150, PT155, PT156,PT157, PT158, TCY1, combinations thereof, and pharmaceuticallyacceptable salts. The rapid, sensitive, and inexpensive test can be usedto determine patients who have non-normal cortisol production ordisordered circadian rhythms as a method for selecting subjects for GCRantagonist or active agent therapy for whom it is likely to havebeneficial and/or therapeutic effects, and can also be used to monitorchanges in cortisol levels in response to treatment.

As used herein, the term “agent” refers to any molecule, compound,methodology and/or substance for use in the prevention, treatment,management and/or diagnosis of viral infection or cancer. Non-limitingexamples of cancer therapies include chemotherapies, radiationtherapies, hormonal therapies, anti-angiogenesis therapies, targetedtherapies, and/or biological therapies including immunotherapies andsurgery.

As used herein, the term “cancer cells” refer to cells that acquire acharacteristic set of functional capabilities during their development,including the ability to evade apoptosis, self-sufficiency in growthsignals, insensitivity to anti-growth signals, tissueinvasion/metastasis, significant growth potential, and/or sustainedangiogenesis. The term “cancer cell” is meant to encompass bothpre-malignant and malignant cancer cells.

As used herein, the term “cancer stem cell(s)” refers to a cell that canbe a progenitor of a highly proliferative cancer cell. A cancer stemcell has the ability to re-grow a tumor as demonstrated by its abilityto form tumors in immunocompromised mice, and typically to form tumorsupon subsequent serial transplantation immunocompromised mice. Cancerstem cells are also typically slow-growing relative to the bulk of atumor; that is, cancer stem cells are generally quiescent. In certainembodiments, but not all, the cancer stem cell may representapproximately 0.1 to 10% of a tumor.

As used herein, the phrase “diagnostic agent” refers to any molecule,compound, and/or substance that is used for the purpose of diagnosing adisease or condition. Non-limiting examples of diagnostic agents includeantibodies, antibody fragments, or other proteins, including thoseconjugated to a detectable agent. As used herein, the term “detectableagents” refer to any molecule, compound and/or substance that isdetectable by any methodology available to one of skill in the art.Non-limiting examples of detectable agents include dyes, gases, metals,or radioisotopes. As used herein, diagnostic agent and “imaging agent”are equivalent terms.

In an embodiment of the invention, the amount of a therapy is effectiveto achieve one, two, three, or more results following the administrationof one, two, three or more therapies: (1) a stabilization, reduction orelimination of the cancer stem cell population; (2) a stabilization,reduction or elimination in the cancer cell population; (3) astabilization or reduction in the growth of a tumor or neoplasm; (4) animpairment in the formation of a tumor; (5) eradication, removal, orcontrol of primary, regional and/or metastatic cancer; (6) a reductionin mortality; (7) an increase in disease-free, relapse-free,progression-free, and/or overall survival, duration, or rate; (8) anincrease in the response rate, the durability of response, or number ofpatients who respond or are in remission; (9) a decrease inhospitalization rate; (10) a decrease in hospitalization lengths; (11)the size of the tumor is maintained and does not increase or increasesby less than 10%, preferably less than 5%, preferably less than 4%,preferably less than 2%; (12) an increase in the number of patients inremission; (13) an increase in the length or duration of remission; (14)a decrease in the recurrence rate of cancer; (15) an increase in thetime to recurrence of cancer; and (16) an amelioration of cancer-relatedsymptoms and/or quality of life.

As used herein, the term “predetermined reference range” refers to areference range for the particular biological entity, e.g., cortisol,for a subject or a population of subjects. Each laboratory may establishits own reference range for each particular assay, or a standardreference range for each assay may be made available and used locally,regionally, nationally, or worldwide or may be patient-specific. In onespecific embodiment, the term refers to a reference range for the amountof cortisol in a patient or a specimen from a patient. In anotherspecific embodiment, the term refers to a reference range for the amountof cortisol in a patient or a specimen from a patient.

As used herein, the term “therapeutic agent” refers to any molecule,compound, and/or substance that is used for the purpose of treatingand/or managing a disease or disorder. Examples of therapeutic agentsinclude, but are not limited to, proteins, immunoglobulins (e.g.,multi-specific Igs, single chain Igs, Ig fragments, polyclonalantibodies and their fragments, monoclonal antibodies and theirfragments), peptides (e.g., peptide receptors, selectins), bindingproteins, biologics, chemospecific agents, chemotoxic agents (e.g.,anti-cancer agents), proliferation-based therapy, radiation,chemotherapy, anti-angiogenic agents, and small molecule drugs.

As used herein, the terms “therapies” and “therapy” can refer to anymethod(s), composition(s), and/or agent(s) that can be used in theprevention, treatment and/or management of a disease or condition, suchas cancer or viral infection, or one or more symptoms thereof. Incertain embodiments, the terms “therapy” and “therapies” refer tochemotherapy, small molecule therapy, radioimmunotherapy, toxin therapy,prodrug-activating enzyme therapy, biologic therapy, antibody therapy,surgical therapy, hormone therapy, immunotherapy, anti-angiogenictherapy, targeted therapy, epigenetic therapy, demethylation therapy, histone deacetylase inhibitor therapy, differentiation therapy, radiationtherapy, or a combination of the foregoing and/or other therapies usefulin the prevention, management and/or treatment of a cancer or one ormore symptoms thereof. As used herein, the terms “treat,” “treatment,”and “treating” in the context of the administration of a therapy to asubject refer to the reduction or inhibition of the progression and/orduration of a disease or condition, such as cancer or viral infection,the reduction or amelioration of the severity of a disease or condition,such as cancer, and/or the amelioration of one or more symptoms thereofresulting from the administration of one or more therapies. In specificembodiments, such terms refer to one, two or three or more resultsfollowing the administration of one, two, three or more therapies: (1) astabilization, reduction or elimination of the cancer stem cellpopulation; (2) a stabilization, reduction or elimination in the cancercell population; (3) a stabilization or reduction in the growth of atumor or neoplasm; (4) an impairment in the formation of a tumor; (5)eradication, removal, or control of primary, regional and/or metastaticcancer; (6) a reduction in mortality; (7) an increase in disease-free,relapse-free, progression-free, and/or overall survival, duration, orrate; (8) an increase in the response rate, the durability of response,or number of patients who respond or are in remission; (9) a decrease inhospitalization rate; (10) a decrease in hospitalization lengths; (11)the size of the tumor is maintained and does not increase or increasesby less than 10%, preferably less than 5%, preferably less than 4%,preferably less than 2%; and (12) an increase in the number of patientsin remission. In certain embodiments, such terms refer to astabilization or reduction in the cancer stem cell population. In someembodiments, such terms refer to a stabilization or reduction in thegrowth of cancer cells. In some embodiments, such terms refer to astabilization or reduction in the cancer stem cell population and areduction in the cancer cell population. In some embodiments, such termsrefer to a stabilization or reduction in the growth and/or formation ofa tumor. In some embodiments, such terms refer to the eradication,removal, or control of primary, regional, or metastatic cancer (e.g.,the minimization or delay of the spread of cancer). In some embodiments,such terms refer to a reduction in mortality and/or an increase insurvival rate of a patient population. In further embodiments, suchterms refer to an increase in the response rate, the durability ofresponse, or number of patients who respond or are in remission. In someembodiments, such terms refer to a decrease in hospitalization rate of apatient population and/or a decrease in hospitalization length for apatient population.

The invention is directed to the use of, for example, a glucocorticoidreceptor antagonist for preventive anti-infective therapy after, forexample, stroke, and for the production of medicines and/orpharmaceutical preparations for preventive anti-infective therapy afterstroke. Respiratory tract infections ranged from 1-33% after stroke andurinary tract infections ranged from 2-27%. These infections have a highimpact on morbidity and mortality. The 30-day-mortality rate in patientswith pneumonia is 27% while the mortality rate is 4% in stroke patientswithout pneumonia. Also, the disability in stroke patients withpneumonia is higher than in patients without, resulting in increasedmedical costs and a lower quality of life. It is ethically and medicallypreferred to prevent or treat the post-stroke infections. Presently,trials of antibiotics for the prevention and treatment of post-strokeinfections are underway.

The term “early preventive, anti-infective therapy after stroke” meansthat the treatment is started within 72 hours after the stroke event.

Glucocorticoid Receptor

The glucocorticoid receptor is widely distributed and expressed in manycultured cell lines, and the control of gene expression byglucocorticoids, therefore, has been widely studied as a model fortranscriptional regulation.

A number of glucocorticoid-responsive transcription units, includingmouse mammary tumor virus (MMTV) (Ringold, et al., 1975; Parks, et al.,1974), mouse and human metallothionein (Eager, et al., 1981; Karin, etal., 1980), rat alpha. sub.2M-globulin (Kurtz, et al., 1977) and rat andhuman growth hormone (Spindler, et al., 1982; Evans, et al., 1982;Robins, et al., 1982) genes have been identified. DNA sequencesmediating transcriptional stimulation of several of these genes havebeen localized. For MMTV, these sequences are discrete genomic regionsupstream of the transcriptional start site which appear to exert theiractions independently of orientation and position (Chandler, et al.,1983; Ostrowski, et al., 1984). The steroid/receptor complex appears tobind to these regulatory sequences and purified receptor has been usedto define the specific binding sites (Govinda, et al., 1982;Scheidereit, et al., 1983; Pfahl, 1982; Payvar, et al., 1983). Theability of the glucocorticoid-responsive element (GRE) to alter itsposition and orientation yet still maintain promoter inducibilitysuggests that it resembles the class of cis-acting regulatory sequencestermed enhancers (Chandler, et al., 1983). First discovered in virusesand subsequently in cellular genes, these sequences are necessary forefficient transcription in vivo (Laimonis, et al., 1982; Benoist, etal., 1981; Baerji, et al., 1983). It has been suggested that enhancersare recognized by trans-acting factors that mediate regulatory effectsby tissue-specific transcriptional control. Although the enhancerfactors have not been well characterized, the glucocorticoid receptormay serve as a paradigm for these putative gene activator proteins.

It is generally accepted that the unliganded glucocorticoid receptor(GR) resides in the cytoplasm, and that hormone activation leads both tonuclear accumulation and gene activation. (Gasc, J.-M. & Baulieu, E. E.(1987) in Steroid Hormone Receptors: Their Intracellular Localisation,ed. Clark, C. R. (Ellis Horwood Ltd., Chichester, England), pp. 233-250;Beato, M. (1989) Cell 56, 335-344; Carson-Jurica, M. A., Schrader, W. T.& OMalley, B. W. (1990) Endocr. Rev. 11, 201-220; Gronemeyer, H. (1993)in Steroid Hormone Action, ed. Parker, M. G. (Oxford University Press,New York), pp. 94-117; Tsai, M. J. & OMalley, B. W. (1994) Annu. Rev.Biochem. 63, 451-486; Akner, G., Wikstrom, A. C. & Gustafsson, J. A.(1995) J. Steroid Biochem. Mol. Biol. 52, 1-16), and references therein.However, the mechanisms involved in nuclear translocation and targetingof steroid receptors to regulatory sites in chromatin have been poorlyunderstood. It has previously been difficult to discriminate between theability of a given receptor mutant, or a given receptor/ligandcombination, to participate in the separate processes of receptoractivation, nuclear translocation, sequence-specific binding, andpromoter activation.

The glucocorticoid receptor (GR) is expressed in a subset of bothERalpha-positive and -negative human breast cancers as well as in othermalignancies such as some ovarian cancers, hepatocellular carcinoma, andesophageal squamous cell carcinoma. In vitro and in vivo experimentssuggest that activation of the GR in ER-negative pre-malignant breastepithelial and cancer cells initiates cell survival pathways understress conditions that normally induce significant cell death (e.g.chemotherapy, radiation, growth factor deprivation).

The glucocorticoid receptor (GR) is present in glucocorticoid responsivecells where it resides in the cytosol in an inactive state until it isstimulated by an agonist. Upon stimulation the glucocorticoid receptortranslocates to the cell nucleus where it specifically interacts withDNA and/or protein(s) and regulates transcription in a glucocorticoidresponsive manner. Two examples of proteins that interact with theglucocorticoid receptor are the transcription factors, API andNFkappa-B. Such interactions result in inhibition of API- andNFkappa-B-mediated transcription and are believed to be responsible forsome of the anti-inflammatory activity of endogenously administeredglucocorticoids. In addition, glucocorticoids may also exert physiologiceffects independent of nuclear transcription. Biologically relevantglucocorticoid receptor agonists include cortisol and corticosterone.Many synthetic glucocorticoid receptor agonists exist includingdexamethasone, prednisone and prednisolone.

Glucocorticoid Receptor Antagonists

Glucocorticoid receptor antagonists bind to the receptor and preventglucocorticoid receptor agonists from binding and eliciting GR mediatedevents, including transcription. RU486 is an example of a non-selectiveglucocorticoid receptor antagonist.

Compounds having high glucocorticoid receptor binding affinity and, inaddition, high in vivo anti-glucocorticoid activity, while having, forexample, low androgenic and progestagenic activities are disclosed inU.S. Pat. No. 6,011,025, incorporated herein by reference in itsentirety. ORG 34517 (PT150) is an example of a compound with highglucocorticoid receptor binding affinity while having low androgenic andprogestagenic activities, show specific and high glucocorticoid receptorbinding affinity and are highly active in vivo showing predominantanti-glucocorticoid activity. The compounds lack appreciable affinityfor mineralocorticoid, progesterone, estrogen and androgen receptors,indicating a clean side effect profile.

ORG 34517, PT150, PT155, PT156, PT157, PT158, TCY1, combinationsthereof, and pharmaceutically acceptable salts thereof derivatives ofthe invention can be used in the prevention and treatment ofglucocorticoid dependent diseases or symptoms, like Cushing syndrome,diabetes, glaucoma, sleep disturbances, depression, anxiety,atherosclerosis, hypertension, adiposity, osteoporosis and withdrawalsymptoms from narcotics and their mixtures.

Preferred compounds according to this invention are ORG 34517, PT150,PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof. Preferred active agentsaccording to this invention are ORG 34517, PT150, PT155, PT156, PT157,PT158, TCY1, combinations thereof, and pharmaceutically acceptable saltsthereof.

GR antagonists from the following structural classes are presented:octahydrophenanthrenes, spirocyclic dihydropyridines, triphenylmethanesand diaryl ethers, chromenes, dibenzyl anilines, dihydroisoquinolines,pyrimidinediones, azadecalins, and aryl pyrazolo azadecalins. Exemplaryglucocorticoid receptor antagonists include, but are not limited to,mifepristone, RU486, 11β-(4-dimethylaminoethoxypheny)-17α-propynyl-17β-hydroxy-4,9 estradien-3-one,17β-hydroxy-17α-19-(4-methylphenyl)androsta-4,9(11)-dien-3-one-4α(S)-Benzyl-2(R)-prop-1-ynyl-1,2,3,4,4α,9,10,10 α (R)-o-ctahydro-phenanthrene-2,7-diol and4.alpha.(S)-Benzyl-2(R)-chloroethynyl-1,2,3,4,4 α,9,10,10α(R)-octahydro-phenanthrene-2,7-diol, and(11β,17β)-11-(1,3-benzodioxo-5-yl)-17-hydroxy-17-(1-propynyl)es-tra-4,9-dien-3-one.Examples include, but are not limited to, beclometasone, betamethasone,budesonide, ciclesonide, flunisolide, fluticasone, mifepri stone,mometasone, and triamcinolone. In further embodiments it may be CORT0113083 or CORT 00112716.

Examples of steroidal glucocorticoid receptor antagonists include,without limitation, mifepri stone, cortexolone, dexamethasone-oxetanone,19-nordeoxycorticosterone, 19-norprogesterone, cortisol-21-mesylate;dexamethasone-21-mesylate,11(-(4-dimethylaminoethoxyphenyl)-17(-propynyl-17(-hydroxy-4,9-estradien-3one,and 17(-hydroxy-17(-19-(4-methylphenyl)androsta-4,9(11)-dien-3-one.

Examples of non-steroidal glucocorticoid receptor antagonists include,without limitation, N-(2-[4,4′,441-trichlorotrityl] oxyethyl)morpholine;1-(2[4,4′,4″-trichlorotrityl]oxyethyl)-4-(2-hydroxyethyl)piperazinedimaleate; N-([4,4′,4″]-trichlorotrityl)imidazole;9-(3-mercapto-1,2,4-triazolyl)-9-phenyl-2,7-difluorofluorenone;1-(2-chlorotrityl)-3,5-dimethylpyrazole;4-(morpholinomethyl)-A-(2-pyridyl)benzhydrol;5-(5-methoxy-2-(N-methylcarbamoyl)-phenyl)dibenzosuberol;N-(2-chlorotrityl)-L-prolinol acetate;1-(2-chlorotrityl)-1,2,4-triazole; 1,S-bis(4,4′,4″-trichlorotrityl)-1,2,4-triazole-3-thiol; 4. alpha.(S)-Benzyl-2(R)-chloroethynyl-1,2,3,4,4.alpha.,9,10,10alpha(R)-octahydro-phenanthrene-2,7-diol (“CP 394531”),4alpha(S)-Benzyl-2(R)-prop-1-ynyl-1,2,3,4,4.alpha.,9,10,10alpha(R)-oc-tahydro-phenanthrene-2,7-diol(“CP-409069”),trans-(1R,2R)-3,4-dichloro-N-methyl-N-[2-1pyrrolidinyl)cyclohexyl]benzene-acetamide,bremazocine, ethylketocyclazocine and naloxone.

The specificity of ORG 34517, PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof forGR blockade, without significant cross-binding to other relatedsteroidal hormone receptors (such as those for estrogen andprogesterone), eliminates the likelihood of significant toxicities andside effects. Indeed, none were identified in all the substantial phaseI and phase II clinical trials that already have been performed with thePT150 compound. Because the drug is envisioned as being used in limiteddosing over time, coordinated with the intermittent dosing strategiestypical for chemotherapeutic agents, the GR blockade also would not leadto significant alteration of HPA-axis functioning, with rapidrestitution of the HPA-axis to baseline following dosing.

The (−)-enantiomer of PT150 (ent-PT150) and of PT155 (ent-PT155) standas being representative for the corresponding unnatural enantiomers ofthe steroid parts in PT155, PT156, PT157 and PT158. We claim theunnatural (−)-enantiomers, and all possible existing steroidaldiastereomers, of the steroid parts in PT155, PT156, PT157and PT158 as asubject of the art.

PT153 is an inclusion complex of one molecule ORG34517 (guest) with twomolecules ORG34517 thiosemicarbazone (host). PT153 is formed byrefluxing ORG34517 in 90% (v/v) aqueous ethanol with an excess ofthiosemicarbazide [H2N—NH—(C═S)—NH2]. The product is precipitated byaddition of water or freezing.In-depth analysis of the reaction product revealed that the productPT153 consisted of two molecules ORG34517 thiosemicarbazone and oneunreacted molecule of ORG34517 in conjunction with the solvents ethanol(1.52×) and water (1.25×) which could not being removed by prolongedvacuum drying over anhydrous calcium chloride (CaCl2):

The ORG34517 thiosemicarbazone part is a cis/trans stereoisomericmixture [(Z/E)-mixture] with 71.8% (E)-stereoisomer (trans) and 28.2%(Z)-stereoisomer (cis):

The proton nuclear magnetic resonance (¹H-NMR) spectrum clearly resolvedthe three molecular species [(E)-thiosemicarbazone,(Z)-thiosemicarbazone, ORG34517] contained in PT153. This compositionwas clearly confirmed by elemental analysis (combustion analysis ofelemental CHNS/O content):

Compound: PT153 Molecular formula: C₂₈H₃₀O₄ × 2C₂₉H₃₃N₃O₃S × 1.52C₂H₅OH× 1.25H₂O Molecular weight: 1530.39 g/mol Elemental analysis:calculated: C 69.88% H 7.09% N 5.49% S 4.19% O 13.35% found: C 69.91% H7.25% N 5.69% S 3.85% O 13.09% C 69.87% H 7.29% N 5.69% S 3.91% O 13.16%

The (E/Z)-stereoisomerism resulting from hindered rotation aboutthioamide groups (H₂N—C═S) is well-known for thiosemicarbazones. Thenature of the inclusion complex will be revealed in the discussion ofthe structure of PT154.

An important feature of PT153 is the strongly electrostatic nature ofits powder form. The powder of PT153 is so inductively magnetic that itposes considerable difficulties in grounding, powdering and dosage. Thepowder jumps from every spatula when grounded. Taken together, thismagnetic nature of PT153 poses considerable difficulties inpharmaceutical formulation. As will be shown, PT154 does not exhibitthis peculiar phenomenon in such intensity.

PT154

PT154 is a purified inclusion complex of one molecule ORG34517 (guest)with two molecules ORG34517 thiosemicarbazone (host).

To synthesize a putative dimer, PT153 was treated with a molar excess ofaqueous sodium hydroxide (NaOH) in acetone at room temperature. Theproduct PT154 was precipitated by addition of water and freezing. Themodel reaction for this experiment was the dimerization of a retinoidthiosemicarbazone published in 2011.

In-depth analysis of the reaction product revealed that the productPT154 consisted of two molecules ORG34517 thiosemicarbazone and oneunreacted molecule of ORG34517 in conjunction with the solvents ethanol(1×), diacetone alcohol (0.24×) and water (0.8×) which could not beingremoved by prolonged vacuum drying over anhydrous calcium chloride(CaCl₂). The diacetone alcohol resulted from the well-knownbase-catalysed dimerization of the reaction solvent acetone [2H₃C—(C═O)—CH₃→(H₃C)₂C(OH)—CH₂—(C═O)—CH₃]. Diacetone alcohol is arelatively non-toxic solvent also found as a natural product in sleepygrass (Achnatherum robustum, Poaceae; syn. Stipa robusta).

The ORG34517 thiosemicarbazone part in PT154 was analyzed to be exactlythe same cis/trans stereoisomeric mixture as in PT153 [(Z/E)-mixturewith 71.8% (E)-stereoisomer (trans) and 28.2% (Z)-stereoisomer (cis)].

The proton nuclear magnetic resonance (¹H-NMR) spectrum clearly resolvedthe three molecular species [(E)-thiosemicarbazone,(Z)-thiosemicarbazone, ORG34517] contained in PT154 (see Experimentalsection). This composition was clearly confirmed by elemental analysis(combustion analysis of elemental CHNS/O content):

Compound: PT154 Molecular formula: C₂₈H₃₀O₄ × 2C₂₉H₃₃N₃O₃S × C₂H₅OH ×0.24C₆H₁₂O₂ × 0.8H₂O Molecular weight: 1526.21 g/mol Elemental analysis:calculated: C 70.39% H 7.03% N 5.51% S 4.20% O 12.87% found: C 70.23% H7.10% N 5.62% S 4.13% O 12.78% C 69.98% H 7.16% N 5.59% S 4.11% O 12.78%

This striking analogy of PT154 to PT153 is remarkable, since completedissolution, treatment with base and precipitation leads almostinvariantly to a change in composition and stereoisomeric proportions.Furthermore, a sodium salt should have been formed from the ORG34517thiosemicarbazone. Nothing of these changes happened at all. Thisstrongly pointed to a complex formation (inclusion compound) of ORG34517in an ORG34517 thiosemicarbazone molecular lattice. The ORG34517thiosemicarbazone exhibits a thiourea grouping, and thiourea iswell-known for its ability to build macromolecular inclusioncompounds⁹⁻¹¹, so-called clathrates. Therefore, it is assumed that PT153and PT154 are, in fact, stable inclusion compounds of one moleculeORG34517 (guest) with two molecules ORG34517 thiosemicarbazone (host).

The reasons for this assumption are (i) the exactly analogouscomposition of PT153 and PT154, despite of PT153's treatment with baseand re-precipitation to yield PT154, (ii) the thiourea structuralelement in PT153 and PT154 enables thiourea-like inclusion compounds asexemplified by the hexamethylenetetramine inclusion complex withthiourea (ratio 1:2)¹¹, and (iii) the existence of steroidal inclusioncompounds as exemplified by cholesterol (guest) with deoxycholate (host)according to Nobel laureate Heinrich Wieland.

The most suitable explanation for the interaction of one moleculeORG34517 (guest) with two molecules ORG34517 thiosemicarbazone (host) isthe published observation of complex formation of testosterone withhippuric acid (or also phenyl urethane, acetanilide, acetamide) (all inratio 1:2). Also deoxycorticosterone (21-hydroxypregn-4-ene-3,20-dione)or dehydro-epi-androsterone 3-O-acetate[(313)-3-(acetyloxy)androst-5-en-17-one] form complexes with hippuricacid (both in ratio 1:2). This means that 3-keto-Δ⁴-steroids likeORG34517 can form addition compounds with amide (orthioamide)-presenting compounds in ratio 1:2.

Very interestingly, thiourea complexes with pyridinium halides(2:1)^(14,15) can exhibit ferroelectric/dielectric properties in thecase of pyridinium iodide/thiourea (1:2)¹⁵. This might be an explanationfor the electrostatic/magnetic nature of PT153.

PT155 PT155: ⅓(11β,17β)-17-Hydroxy-11-[3,4-(methylenedioxy)phenyl]-17-(1-propyn-1-yl)estra-4,9-dien-3-one(ORG34517)×(2EZ)-2-{(11β,17β)-17-hydroxy-11-[3,4-(methylenedioxy)phenyl]-17-(1-propyn-1-yl)estra-4,9-dien-3-ylidene}hydrazinecarbothioamide[71.8% (E), 28.2% (Z)] hemihydrate×¾ acetone×⅛ ethanol (PT155)

PT155 is an inclusion complex of choice of, for example, one moleculeORG34517 (guest) on three molecules ORG34517 thiosemicarbazone (host).In exemplary embodiments, the ratio of ORG34517 thiosemicarbazone toORG34517 is, for example, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1.

Compound: PT155 Molecular formula: ⅓C₂₈H₃₀O₄ × C₂₉H₃₃N₃O₃S ×¾C₃H₅O ×⅛C₂H₅OH × ½H₂O Molecular weight: 705.49 g/mol Elemental analysis:calculated: C 69.52% H 7.04% N 5.96% S 4.55% O 12.95% found: C 69.81% H7.17% N 5.82% S 4.25% O 12.90% C 69.40% H 7.13% N 5.91% S 4.48% O 12.70%

In an experiment aimed at forcing the reaction in direction of ORG34517thiosemicarbazone, the reflux time was doubled from 30 min to 1 h. Onlya similar inclusion complex as contained in PT153 and PT154 could beisolated. It is concluded that ORG34517 thiosemicarbazone and ORG34517interact strongly as previously postulated. The ORG34517thiosemicarbazone part of PT155 is a (Z/E)-mixture with 71.8%(E)-stereoisomer and 28.2% (Z)-stereoisomer. This material PT155 is thecomplex of choice to be used in antiviral studies in vitro. Themetabolic activation of ORG34517 thiosemicarbazone contained inPT153-155:

S-Oxidation by Human Flavin-Containing Monooxygenases (hFMOs).

It is well-known that thiosemicarbazones like thiacetazone(p-acetamidobenzaldehyde thiosemicarbazone), a cheap second-lineantitubercular substance discovered by Nobel laureate Gerhard Domagk in1946, are bio-activated by human flavin-containing monooxygenases(hFMO1, hFMO2.1, hFMO3) into a sulfenic acid (R—S—OH), a sulfinic acid[R—(S═O)—OH], and a carbodiimide derivative (R—N═C═N—H)23,24:

Both the sulfenic acid and the carbodiimide derivative are the activeantitubercular metabolites. The sulfinic acid (bottom left) does notform the carbodiimide, and represents an inactive metabolite. Especiallythe carbodiimide derivative can react with amino acid residues in targetproteins of, for example, Mycobacterium tuberculosis, or with humanhepatic/extrahepatic glutathione R—SH thiol/mercapto group for metabolicdetoxification:

Therefore, ORG34517 thiosemicarbazone will be bio-activated (S-oxidized)in the same manner as thiacetazone by hFMOs under consumption ofmolecular oxygen (02), and will be able to bind covalently to HBV andHIV proviral GRE DNA after being shuttled to the host cell nucleus byhGR. The thiosemicarbazone moiety in ORG34517 thiosemicarbazone can beoxidized by hFMOs (hFMO1, hFMO2.1, hFMO3) with molecular oxygen over thesulfenic acid to the ORG34517 thiosemicarbazone carbodiimide which couldbe co-transported into the host cell nucleus by hGR: hGRsubunit1-(ORG34517 thiosemicarbazone carbodiimide)=hGRsubunit 2-ORG34517. Thecarbodiimide moiety adds to one amino group of proviral GRE DNA yieldingan N-amidinohydrazone (amidrazone)-group, thereby inducing proviral DNAdamage.

Human flavin-dependent monooxygenases (hFMOs) (EC 1.14.13.8) are thesecond important type of monooxygenases in the human body, the otherwell-known type being the cytochrome P450 monooxygenases (CYP450monooxygenases). Both are localized in microsomes and dependent onmolecular oxygen (02), and hFMOs need also the cofactors flavin-adeninedinucleotide (FAD) and nicotinamide-adenine dinucleotide phosphate(NADPH), whereas CYP450 monooxygenases are heme-dependent. hFMO1 ismainly expressed in human kidney, and, to a smaller extent, in smallintestine and lung. hFMO2 is expressed to a very high level in humanlung and kidney, and, to a smaller extent, in liver and smallintestine28. hFMO3 and hFMO5 are highly expressed in human liver, butare also expressed in lung and, to a smaller extent, in human kidney.hFMO4 is mainly expressed in kidney and, to a smaller extent, in liverand small intestine. hFMO5 is also expressed to a high level in smallintestine. FMO2 is additionally expressed in human brain, but at lowabundance (<1% of lung).

Slightly modified citation: “The hFMOs oxygenate nucleophilicheteroatom-containing chemicals and drugs and generally converts theminto harmless, polar, readily excreted metabolites. Sometimes, however,FMO bioactivates chemicals into reactive materials that can causetoxicity. Most of the interindividual differences of hFMOs are due togenetic variability and allelic variation, and splicing variants maycontribute to interindividual and interethnic variability observed forhFMO-mediated metabolism. In contrast to cytochrome P450 monooxygenases(CYP450 monooxygenases), hFMOs are not easily induced nor readilyinhibited, and potential adverse drug-drug interactions are minimizedfor drugs prominently metabolized by hFMOs. These properties may provideadvantages in drug design and discovery, and by incorporating hFMOdetoxication pathways into drug candidates, more drug-like materials maybe forthcoming. Although exhaustive examples are not available,physiological factors can influence hFMO function, and this may haveimplications for the clinical significance of hFMOs and a role in humandisease.”

PT155 exhibits magnetism is of electrostatic type, and is called thetriboelectric effect. The triboelectric effect (also known astriboelectric charging) is a type of contact electrification in whichcertain materials become electrically charged after they come intofrictional contact with a different material. Rubbing glass with fur, ora comb through the hair, can build up triboelectricity. Most everydaystatic electricity is triboelectric. The polarity and strength of thecharges produced differ according to the materials, surface roughness,temperature, strain, and other properties.

PT-156 PT156:1/17(11β,17β)-17-Hydroxy-11-[3,4-(methylenedioxy)phenyl]-17-(1-propyn-1-yl)estra-4,9-dien-3-one(ORG34517)×(2EZ)-2-{(11β,17β)-17-hydroxy-11-[3,4-(methylenedioxy)phenyl]-17-(1-propyn-1-yl)estra-4,9-dien-3-ylidene}-N-phenylhydrazinecarbothioamide[74.1% (E), 25.9% (Z)] 11/17 hydrate (PT156) PT156: ORG345174-phenylthiosemicarbazone containing traces of ORG34517

In exemplary embodiments, PT156 is(2EZ)-2-{(11β,17β)-17-hydroxy-11-[3,4-(methylenedioxy)phenyl]-17-(1-propyn-1-yl)estra-4,9-dien-3-ylidene}-N-phenylhydrazinecarbothioamidewithout the presence of(11β,17β)-17-Hydroxy-11-[3,4-(methylenedioxy)phenyl]-17-(1-propyn-1-yl)estra-4,9-dien-3-one(ORG34517). In exemplary embodiments, the ratio of PT156 to ORG34517 is30:1, 20:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1,7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1.

The elemental analysis confirmed the results obtained by 1H NMRspectroscopy:

Compound: PT156 Molecular formula: 1/17C₂₈H₃₀O₄ × C₂₉H₃₇N₃O₃S × 11/17H₂OMolecular weight: 616.73 g/mol Elemental analysis: calculated: C 71.37%H 6.55% N 6.81% S 6.20% O 10.07% found: C 71.47% H 6.83% N 6.83% S 6.58%O 10.05% C 71.18% H 6.88% N 6.87% S 6.46% O 10.07%

PT156 is formed by refluxing ORG34517 in 90% (v/v) aqueous ethanol witha slight excess of 4-phenylthiosemicarbazide [H2N—NH—(C═S)—NH—C6H5]. Theproduct is precipitated by addition of water and freezing. Analysis ofthe reaction product revealed that the present invention PT156 contained17 1 of an unreacted molecule of ORG34517 in conjunction with thesolvent water (17 11×) which could not being removed by prolonged vacuumdrying over anhydrous calcium chloride (CaCl2). The ORG345174-phenylthiosemicarbazone part is a cis/trans stereoisomeric mixture[(Z/E)-mixture] with 74.1% (E)-stereoisomer (trans) and 25.9%(Z)-stereoisomer (cis).

The Metabolic Activation of ORG34517 4-Phenylthiosemicarbazone Containedin PT156: Para-Oxidation by Human Cytochrome P450 Monoxygenases

It is well-known that acetanilide is oxidized by human cytochrome P450monooxygenase 1A2 (CYP1A2) isoenzyme in para-position of the phenyl ringto yield paracetamol (acetaminophen)29, and is further oxidized byCYP3A4 to N-acetyl-p-benzoquinone imine:

Analogously, PT156 could be activated by CYP1A2 and CYP3A4 in Tlymphocytes to:

Analogously, PT156 could be activated by CYP1A2 and CYP3A4 in Tlymphocytes to: This represents an additional activation mechanism forPT156 which is not achieved with and incorporated in PT155. It isespecially important for the potential treatment of acquiredimmunodeficiency syndrome (AIDS) with PT compounds, since in human bloodlymphocytes only very low levels of human flavin-containingmonooxygenases (only hFMO4 and hFMO5) could be detected. Even in absenceof human flavin-containing monooxygenases PT156 could be activated inhuman T lymphocytes, since microsomal cytochrome P450 isoenzymes 1A2 and3A4 were readily detected in human blood lymphocytes. Historically, theoverall metabolism of acetanilide was elucidated by Brodie & Axelrod in1948.

PT-157

⅓(11β,17β)-17-Hydroxy-11-[3,4-(methylenedioxy)phenyl]-17-(1-propyn-1-yl)estra-4,9-dien-3-one-(2)-2-{(11β,17β)-17-hydroxy-11-[3,4-(methylenedioxy)phenyl]-17-(1-propyn-1-yl)estra-4,9-dien-3-ylidene}hydrazinecarbothioamide[71.8% (E), 28.2%(Z)]-bis[6-amino-2-oxo-3-(β-D-ribofuranosyl)-2,3-dihydropyrimidin-1-ium-1-yl]methanediidemonohydrochloride×½ acetone (PT158)

In exemplary embodiments, PT-158 is:

Materials:

PT155 (C28H30O4×C29H33N3O3 S×¾ C3H6O×C2H5OH×½ H₂O) [Pop Test OncologyLLC, Cliffside Park, N.J., USA; first batch synthesized by Andreas J.Kesel at Thursday, October 8th, 2015; w (n/n). 99% (1H NMR and elementalanalysis)]

TCY-1 (C19H26N6010×HCl×1.25 H2O) [synthesized by Andreas J. Kesel atSaturday, Dec. 26, 2015; w (n/n). 99% (1H NMR and elementalanalysis)]=bis(cytidin-3-ium-3-yl)methanediide monohydrochloride×1.25H₂O=bis[6-amino-2-oxo-3-(â-D-ribofuranosyl)-2,3-dihydropyrimidin-1-ium-1-yl]methanediidemonohydrochloride×1.25 H₂O

Instruction:

PT155 (M=705.49 g/mol, 151 mg, 214.0356 μmol) and TCY-1 (M=557.42 g/mol,121 mg, 217.0715 μmol) (this complete mixture had M=1270.82 g/mol beforedrying) were carefully weighed and thoroughly mixed as solid powderswith a spatula. The mixture was then carefully dried over CaCl2 in vacuoto yield PT158 as light yellow amorphous powder.

-   Compound: PT158-   Molecular formula: (C28H3004×C29H33N3O3S)×(C19H26N6010×HCl)×½ C3H60-   Molecular weight: 1211.11 g/mol-   Yield: 260 mg (100%)

1H-NMR: (DMSO-d6, ppm) 0.41 (3H, s; 18-CH3, (E)-TSC*), 0.42 (1.18H, s;18-CH3, (Z)-TSC**), 0.44 (1.393H, s; 18-CH3, ORG34517), 1.20-2.77 (m;steroid CH and CH2), 1.83 (5.58H, br s; R—C≡C—CH3 methyl, all threespecies), 2.09 (4.18H, s; acetone CH3), 3.57 (dd, 2H; 2J=−11.9 Hz,3J=2.2 Hz; H-5′, pro-R, TCY-1), 3.68 (dd, not resolved, 2H; 2J=−12.2 Hz;H-5′, pro-S, TCY-1), 3.86 (dt, 2H; 3J=5.6 Hz, 3J=2.6 Hz; H-4′, TCY-1),3.95 (t, 2H; 3J=5.1 Hz; H-3′, TCY-1), 4.00 (t, 2H, 3J=4.5 Hz; H-2′,TCY-1), 4.28 (0.393H, m; 11â-CH, (Z)-TSC), 4.30 (1H, d; 3J (H, H)=7.1Hz; 11â—CH, (E)-TSC), 4.38 (0.464H, d; 3J (H, H)=7.1 Hz; 11â—CH,ORG34517), 5.08-5.16 (1.857H+4H, br m; 17â-OH, all three species; 3′-OH,5′-OH, TCY-1), 5.42 (br s, 2H; 2′-OH, TCY-1), 5.66 (0.464H, s; 4-CH,ORG34517), 5.74 (d, 2H; 3J=3.8 Hz; H-1′, TCY-1), 5.86 (1H, s; 4-CH,(E)-TSC), 5.94 (d, 2H; 3J=7.7 Hz; H-5, TCY-1), 5.97 (3.716H, br s;O—CH2-O benzodioxole, all three species), 5.97 (0.393H, s; 4-CH,(Z)-TSC), 6.60 (1.858H, d; 3J(H, H)=7.7 Hz; 5′-CH benzodioxole, allthree species), 6.67 (0.393H, s; 2′-CH benzodioxole, (Z)-TSC), 6.77(1.464H, s; 2′-CH benzodioxole, (E)-TSC and ORG34517), 6.79 (0.393H, m;6′-CH benzodioxole, (Z)-TSC), 6.79 (1.464H, d; 3J(H, H)=8.3 Hz; 6′-CHbenzodioxole, (E)-TSC and ORG34517), 7.51 (0.393H, br s; NH2, HA,(Z)-TSC), 7.57 (1H, br s; NH2, HA, (E)-TSC), 7.92 (br s, 2H; 4-NH2, HA,TCY-1), 7.97 (0.393H, br s; NH2, HB, (Z)-TSC), 8.08 (1H, br s; NH2, HB,(E)-TSC), 8.09 (d, 2H; 3J=7.7 Hz; H-6, TCY-1), 8.48 (br s, 2H; 4-NH2,HB, TCY-1), 10.05 (1H, br s; N—H, (E)-TSC), 10.42 (0.393H, br s; N—H,(Z)-TSC), 13.00 (br s, 1H; hydrochloride, TCY-1). *,**(E or Z)-TSC=(E orZ)-thiosemicarbazone.

TCY-1 Bis(cytidin-3-ium-3-yl)methanediide monohydrochloride×1.25H2O=bis[6-amino-2-oxo-3-(β-D-ribofuranosyl)-2,3-dihydropyrimidin-1-ium-1-yl]methanediidemonohydrochloride×1.25 H2O (TCY-1)

In exemplary embodiments, TCY1 is:

In exemplary embodiments, TCY1 is an HIV Integrase inhibitor. HIVintegrase is an attractive target for the discovery of new therapeuticsdue to its important role in viral infections, particularly HIVinfections. The compounds of the present invention exhibit advantagesover previously disclosed integrase inhibitors, for example increasedpotency, metabolic stability, increased therapeutic index, or otherpharmaceutical properties.

The methods, compounds, compositions, and uses described herein can bespecifically directed to inhibiting HIV integrase in a patient in needthereof. Such methods and uses may prevent, treat or delay the onset ofAIDS in a mammal in need thereof. The present invention also includes acompound of the present invention described herein, and/or apharmaceutically-acceptable salt, hydrate, solvate, tautomer, opticalisomer, E-isomer, Z-isomer, or combination thereof for use in, for useas a medicament for, and/or for use in the preparation of a medicamentfor: inhibiting HIV integrase, preventing or treating infection by HIV,or preventing, treating or delaying the onset of AIDS.

The compounds of the present invention may also be used with one or moreagents useful in the treatment of HIV infection or AIDS. As thecompounds of the present invention can be HIV integrase inhibitors, suchcompounds are also useful in salvage therapy for patients whose virushas mutated and acquired resistance to other drugs. Such inhibitorstarget a distinct step in the retroviral life cycle and therefore, maybe taken in combination with other types of HIV drugs to minimizeadaptation by the virus.

Materials:

Cytidine [Sigma-Aldrich Corp., St. Louis, Mo., USA, Lot: BCBN7660V; w(n/n)=99.9% (HPLC, area %), =+29.7° (c=9 in H2O), =+33.0° (c=2 in H2O),mp 210-220° C. (dec.)] 20D][a20D][a

Thymol (5-methyl-2-isopropylphenol) cryst. Ph. Eur. 1997 [Caesar &Loretz (Caelo) GmbH, Hilden, Germany, Lot: 24252173; residue afterevaporation <0.05% (m/m)]

3% (m/m) aqueous dihydrogen peroxide (H2O2) solution [according toDAC/NRF (Deutscher Arzneimittel-Codex/Neues Rezeptur-Formularium), NRFmonograph No 11.103] stabilized with aqueous ortho-phosphoric acid(H3PO4) [0.0588% (m/m) of 85% (m/m) aqueous H3PO4; this corresponds to0.05% (m/m) H3PO4 final concentration in the stabilized H2O2 solution(this solution showed pH 5.0 at.=19.8° C.)]

10.27 M [32% (m/m)] aqueous hydrochloric acid pro analysi [AppliChem,Darmstadt, Germany, Lot: 3A001639; w (m/m)=33.09% (titration),bromide<0.005%, phosphate<0.00005%, sulfate<0.0001%, As<0.000001%,Fe<0.00002%, heavy metals (Ni, Pb, Zn)<0.000005%]

Ethyl acetate pro analysi [AppliChem GmbH, Lot: 0000518022; w(n/n)=99.9% (GC), w (H₂O)=0.01% (m/m) (Karl Fischer titration),ethanol<0.1%, methanol<0.02%, methyl acetate<0.02%, trace elements (Cr,Fe, Ni, Pb, Zn, P, S, K, Mg)<0.00001%, Si<0.00002%, Na<0.0002%,non-volatile matter<0.001%, acidity/alkalinity<0.0005 meq/g]

Instruction:

Cytidine (M=243.22 g/mol, 10.355 g, 42.5746 mmol), the carbon sourcethymol Ph. Eur. 1997 (M=150.22 g/mol, 6.228 g, 41.4592 mmol), and sodiumhydrogen carbonate NaHCO₃(3.646 g, 43.3996 mmol) were suspended in 90%(v/v) aqueous ethanol (100 ml). Then 3% (m/m) aqueous dihydrogenperoxide (H₂O₂) solution [48 ml, 1.440 g H2O2 (M=34.01 g/mol) 42.3405mmol] was added at room temperature (RT, l=15.6° C.). Solid sodiumhydroxide NaOH (2.064 g, 51.6000 mmol) pearls, and water (80 ml), wereadded under stirring. The suspension became light purple during 10 minstirring at RT. Afterwards, the suspension was heated to 40-50° C. for 5min (heatgun) until all solids had dissolved. The solution was leftstanding at RT for 5 min. Afterwards, the reaction was stopped byaddition of 10.27 M [32% (m/m)] aqueous hydrochloric acid pro analysi(9.20 ml, 94.4840 mmol). The color changed from purple to light yellow.A floating yellow oil evolved soon. The solution (pH 4-5) with thefloating yellow oil was cooled at +0-2° C. for 2 h. The mixture was thenfrozen at −25° C. for 2.5 h. Then sodium hydroxide (520 mg, 13.0000mmol) dissolved in water (3 ml) was added (color change to purple). Themixture was then frozen at −25° C. for 30 min. Then 10.27 M [32% (m/m)]aqueous hydrochloric acid pro analysi (3.00 ml, 30.8100 mmol) was addedunder stirring (color change to yellow). The mixture was then frozen at−25° C. for 105 min. Then sodium hydroxide (740 mg, 18.5000 mmol)dissolved in water (5 ml) was added. The yellow solution was extractedwith ethyl acetate pro analysi (EtOAc, 100 ml). The aqueous phase wasisolated and frozen at −25° C. for 55 h (2 days 7 h). The evolved yield(1.932 g) of the white, crystalline, very odorous (caseous) product wasfiltered and dried over CaCl2 in vacuo.

-   Compound: TCY-1-   Molecular formula: C19H26N6010×HCl×1.25 H₂O-   Molecular weight: 557.42 g/mol-   Yield: 1.932 g (16%)-   Elemental analysis: calculated: C, 40.94% H, 5.33% N, 15.08% O,    32.29%-   found: C, 39.31% H, 5.56% N, 15.26% O, 32.08% C, 39.43% H, 5.63% N,    15.16% O, 32.04%.

1H-NMR: (DMSO-d6, ppm) 3.57 (dd, 2H; 2J=−12.2 Hz, 3J=3.2 Hz; H-5′,pro-R), 3.68 (dd, 2H; 2J=−12.2 Hz, 3J=3.2 Hz; H-5′, pro-S), 3.86 (dt,2H; 3J=5.4 Hz, 3J=3.0 Hz; H-4′), 3.95 (t, 2H; 3J=5.1 Hz; H-3′), 3.99 (m,2H; H-2′), 5.07 (br s, 2H; 3′-OH), 5.15 (br s, 2H; 5′-OH), 5.41 (br s,2H; 2′-OH), 5.74 (d, 2H; 3J=3.8 Hz; H-1′), 5.93 (d, 2H; 3J=7.7 Hz; H-5),7.89 (br s, 2H; 4-NH2, HA),

TABLE 1 The antiretroviral activity of TCY-1 versus HIV-1 strain LAIreplication in PBMC: Anti-HIV-1_(LAI) activity EC₅₀ (μM)/ CytotoxicityCC₅₀ (μM) EC₉₀ (μM) in PBMC Drug PBMC CCRF-CEM Vero EC₅₀ EC₉₀ SI₅₀ r²TCY-1 >100 >100 >100 0.29 1.00 >345 0.94 AZT* >100 14.3 56.0 0.0044 ±0.0039 0.0299 ± 0.0245 >22,696 0.98

Legend:

PBMC, primary human peripheral blood mononuclear cells. CCRF-CEM, humanT-lymphoblastic acute T cell leukemia cells. Vero, African green monkey(grivet) Chlorocebus aethiops (syn. Cercopithecus aethiops) kidneyepithelial cells. CC₅₀, cytotoxic concentration 50%. EC₅₀, effectiveinhibitory concentration 50%. EC₉₀, effective inhibitory concentration90%. SI₅₀, selectivity index CC₅₀/EC₅₀. r², coefficient of determination(r² measure of goodness-of-fit) on EC₅₀ and EC₀₀. AZT, zidovudine(3′-azido-3′-deoxythymidine).

The given effective inhibitory concentrations (μM±s.d.) for the positivecontrol AZT were averaged and treated statistically from twenty (n=20)independent determinations.

Method of Determination: HIV-1 Replication Reverse Transcriptase (RT)Assay with TCY-1HIV-1_(LAI) (=HIV-1_(BRU)=LAV-1; for the origin and identity ofHIV-1_(LAI) was assayed in primary [freshly donated from healthy (testedHIV-negative, HBV-negative, and HCV-negative) blood donors, and isolatedby single-step Ficoll-Hypaque centrifugation method] human peripheralblood mononuclear cells (PBMC) in the presence of a drug beingevaluated. The parameter for antiviral activity was reduction of RTactivity in the cell supernatant after Triton X-100-mediated lysis ofreleased virions, as measured by [5alpha-³H]dTTP (5alpha-tritiatedthymidine 5′-triphosphate) incorporation into poly(rA).poly(dT) directedby the primed RNA template poly(rA).oligo(dT). It should be noted thatthe assay did not detect RT inhibition by potential RT inhibitors perse, but indirectly quantified the amount of released HIV-1 in thesupernatant. The detailed assay methodology was reported by Schinazi etal., as based on an older assay system of Spira et al. The experimentswere conducted in triplicate and treated statistically by regressioncurve analysis (r² coefficient of determination). The RT inhibitor AZT(zidovudine, 3′-azido-3′-deoxythymidine; RETROVIR™) served as a positivecontrol. Cytotoxicity on PBMC exerted by the test compounds wasdetermined as described by Stuyver et al., by application of theCellTiter 96® AQ_(ueous) One Solution Cell Proliferation Assay (PromegaCorp., Madison, Wis.). Briefly, the phenazine ethosulfate (PES)-coupledreduction of the tetrazolium salt3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) to a purple, water-soluble formazan by living, undamaged cells wasmeasured.

Antiviral Activity

PT150, formerly known as ORG34517, was first developed by Organon as adrug therapy for major depressive disorder (Gallagher & Young, 2006). In2007, it was acquired by Schering-Plough, then by Merck & Co., andultimately was acquired by Pop Test Cortisol LLC in December of 2010. AsORG34517, it has undergone two Phases of Clinical Trials for safety:

ClinicalTrials.gov ID NCT00226278 Safety Study of ORG 34517 for MajorDepression (Phase I) With Psychotic Features NCT00844922 Safety of Org34517 900 mg in Patients Who Received (Phase II) Org 34517 in a PreviousTrial (Study 28133/P05842)PT155 was developed from PT150 based on observations thatthiosemicarbazones exhibit considerable antiviral activities, includingactivities versus hepatitis C virus and Ebola virus Zaire (Kesel, 2011;Kesel et al. 2014).

Mechanism of Action

The 3′ UTR is critical to viral replication in the host and interactswith a large number of host proteins (Roby, 2014) including Mov34, whichhas been demonstrated to be involved in transcription and translation inmouse (Ta & Vrati, 2000). The PMSD7 gene product (a Mov34 related gene)is a subunit in the proteasome. Mov34 has been demonstrated to bind tothe genome of the Japanese Encephalitis Virus (Ta & Vrati, 2000).Alterations in the proteasomal processing of NF-κB is a major regulatorypoint for the production of pro-inflammatory cytokines and incontrolling the immune response; microbial and viral pathogens have beendocumented to alter this pathway to promote infection (Rahman &McFadden, 2011). Additionally, Mov34 contains an MPN domain, which ishighly conserved in eukaryotic initiation factors in the 3A family(Asano, 1997; Sanches, 2007).

Without being bound to any theory, one possible mechanism of action forcompounds PT150 and PT155 is rooted in their activity as glucocorticoidreceptor (GR) antagonists. The GR agonist dexamethasone and the GRantagonist mifepristone (RU38486) have been demonstrated to interactwith Mov34 in HIV-1 infection (Ramanathan et al., 2002) and eitherpromote or inhibit translocation to the nucleus. The disruption ofinteractions between Mov34-like proteins and the 3′-UTR region of Zika,and likely other Flavivirus, suppress viral replication and negatesfRNA-mediated cellular events necessary for successful viral infection.This hypothesis is further supported by the patents WO 2004/112720 A2 NDUS 2007/0259844 A1 (Kim 2004; Kim, 2007) for antiviral compositionsagainst the human hepatitis C virus.

Results Justification of Readiness Level

PT150 is a new class of therapeutic agents designed to block theglutocorticoid receptor (GR), acting as an antagonist for endogenouscortisol. PT150 or(11β,17β)-11-(1,3-benzodioxol-5-yl)-17-hydroxy-17-(1-propynyl)-estra-4,9-dien-3-oneis the first selective GR antagonist studied in a Phase I program inhealthy human subjects (ClinicalTrials.gov, NCT00226278) for its safety,tolerability and pharmacodynamic/pharmacokinetic characteristics. Itshowed no prohibitive effects in the first in-human studies. It hassignificantly higher selectivity for the GR compared to mifeprestone(RU486). PT150 and mifepri stone bound to cytosolic GRs with values of365% and 193% respectively; values for the cytosolic progesteronereceptors (PR), however, were 6.4% and 36% respectively. This suggeststhat PT150 has a selectivity ratio (GR/PR) of 57 compared to 5.4 formifeprestone. In the pregnancy interruption test in rats, twice dailyadministration of 4 mg kg⁻¹ po PT150 and 1-2 mg kg⁻¹ po mifepristoneproduced similar results (nearly 100% embryos lost) confirming the loweranti-progestational activity of PT150 as compared to mifeprestone.Therefore, the specificity of PT150 for GR-blockade, without significantcross binding to other related steroidal hormone receptors (such asthose for estrogen and, discretely, progesterone) eliminate thelikelihood of significant toxicities and side effects. In contrast tomifeprestone, PT150 was able to block corticosterone-induced GRtranslocation. This suggests that PT150 is a true competitive GRantagonist without partial agonistic activities (Peeters et al., 2008).

GR expression has not shown prognostic value in Kaplan-Meier survivaland residual survival analysis or overall survival in ovarian cancerpatients (Woenchhaus et al., 2006). Nonetheless, administration of GCalong with apoptosis-inducing chemotherapies to ovarian cancer patientsinhibited cell death and activation of anti-apoptotic genes SGK1,MKP1/DUSP1, and caspase inhibitor cIAP2 in ovarian tissues, suggestingan overall decrease in the effectiveness of chemotherapy (Runnebaum &Bruning, 2005; Melhem et al., 2009).

Results from Viral Testing

The following virus taken from the NIAID Merging Pathogens List areFlavivirus which PT150 and PT155 have the potential to treat.

Problems with Obscured Activity in Existing Test Results

Test results to date have two identified problems that we believe areobscuring antiviral results. Firstly, many of the cell lines used intesting are immortal cell lines. Secondly, not all of the cell linestested contain active glucocorticoid receptors, which play a role incompound interaction for antiviral activity.

Phase II Clinical of PT150 dosed patients at 900 mg per day for 2 weekswith no noted adverse indications of cell toxicity. Yet, cytotoxicity isshowing up for in vitro models. PT discovered that PT150 has value as achemosensitizing agent in the treatment of cancer. As such, it has ademonstrable effect on cancerous cells. Unfortunately, this includesmost cell lines utilized for in vitro testing.

Take, for example, the observable differences in cytotoxicity when PT150is used in the Zika assay in HeLa cells (breast tumor derived cellline), HUH7 (liver tumor cell line) and HFF (human foreskin fibroblasts,non-tumor derived). The 50% cellular toxicity in primary human foreskinfibroblasts is far greater than 150 μM, these cells are very sensitivesensors of toxicity on human tissue.

Vero-derived cell lines (Vero, Vero 76, Vero E6) do not contain humanglucocorticoid receptors, as they are derived from African green monkey(Chlorocebus aethiops) epithelial kidney cells. They even do not evencontain a Chlorocebus glucocorticoid receptor (Dreyer et al., 1989), andhave been demonstrated to be completely unresponsive to dexamethasone.As such, seeing no activity on an antiviral assay for Flavivirus is notsurprising. We believe the choice of cell line obscured potentialpositive results for PT150 and PT155 assays. Re-assay in a cell linethat contains a glucocorticoid receptor (preferably a human one) islikely to yield compound activity

Sexual Transmission of the Zika Virus

The Zika virus has been demonstrated to be sexually transmissiblethrough semen in multiple studies. The RNA virus can be detected insemen up to 62 days after the beginning of infection. (Hill et al.,2016; Mansuy et al., 2016; McCarthy, 2016). This provides not only aninfection route for the virus which is independent of an arthropodvector but also exceeds the current WHO guidelines for engaging inprotected sex for 30 days after returning from an endemically infectedarea (Turmel et al., 2016).

The persistence of Zika in semen offers an opportunity to conductclinical testing in an arena which avoids the issues of birth defectswith testing in pregnant women or women seeking to become pregnant.

ORG34517, PT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof,and pharmaceutically acceptable salts thereof and other GCR-antagonistmolecules necessarily thereby also interfere with the functions of viralGRE and can be used in accord with the following methods: Administrationof ORG34517, PT150, PT155, PT156, PT157, PT158, TCY1, combinationsthereof, and pharmaceutically acceptable salts thereof or otherGCR-antagonist molecules prior to or during physical, social, emotionalor psychological conditions of stress that lead to elevations ofcirculating cortisol in order to diminish or prevent reactivation oflatent viral infections;

Administration of ORG34517, PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof orother GCR-antagonist molecules prior to or during physical, social,emotional or psychological conditions of stress that lead to elevationsof circulating cortisol in order to diminish or prevent susceptibilityto viral infection; Administration of ORG34517, PT150, PT155, PT156,PT157, PT158, TCY1, combinations thereof, and pharmaceuticallyacceptable salts thereof or other GCR-antagonist molecules inindividuals prior to or receiving therapeutic doses of glucocorticoidsin order to diminish or prevent reactivation of latent viral infections;

Administration of ORG34517, PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof orother GCR-antagonist molecules in individuals prior to or receivingtherapeutic doses of glucocorticoids in to diminish or preventsusceptibility to viral infection.

Administration of ORG34517, PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof orother GCR-antagonist molecules in individuals during reactivation oflatent viral infections to diminish intensity of viral reactivation, todiminish length of viral reactivation, to speed time to resolution andhealing of viral reactivation, to speed time to suppression of viralreactivation, to increase likelihood of viral eradication, and/or todiminish infectivity of viral reactivation;

Administration of ORG34517, PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof orother GCR-antagonist molecules in individuals during acute viralinfections to diminish intensity of viral infection, to diminish lengthof viral infection, to speed time to resolution and healing of viralinfection, to speed time to suppression of viral infection, to increaselikelihood of viral eradication, and/or to diminish infectivity of viralinfection.

Administration of ORG34517, PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof orother GCR-antagonist molecules prior to or during travel to environmentsin which viruses are endemic in order to diminish or preventsusceptibility to viral infection

Through interference with the functions of viral GRE, ORG34517, PT150,PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof and other GCR-antagonistmolecules can prevent or reduce tissue damage in forms such as, but notlimited to direct viral cytotoxicity, immune mediate destruction ofvirally infected cells and tissues, and malignant transformation.

ORG34517, PT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof,and pharmaceutically acceptable salts thereof and other GCR-antagonistmolecules may be combined with other, virus-targeted anti-viralmedications (i.e. molecules that specifically bind to viral moleculesand interfere with virus physiology) in the setting of acute infection,reactivation of latent viral infection or active chronic viralinfection.

ORG34517, PT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof,and pharmaceutically acceptable salts thereof and other GCR-antagonistmolecules may be combined with other, non-specific anti-virusmedications (i.e. immune system modulators that work non-specifically tomodulate virus physiology) in the setting of acute infection,reactivation of latent viral infection or active chronic viralinfection.

Viral Conditions

Virus families that infect humans (some of which also infect animals)include: Adenoviridae, Papillomaviridae, Polyomaviridae, Herpesviridaeand Poxviridae. These include, but are not limited to: adenovirus,herpes simplex-1, herpes simplex-2, Varicella-zoster, Epstein-Barrvirus, cytomegalovirus, human herpes virus 8, human papilloma virus, BKvirus, JC virus, chicken pox and small pox.

Hepadnaviridae, including, but not limited to hepatitis B virus.

Parvoviridae, including but not limited to human bocavirus andparvovirus B19.Astroviridae, Caliciviridae, Picornaviridae, Coronoviridae,Flaviviridae, Retroviridae, Togaviridae, Hepeviridae. These include, butare not limited to human astrovirus, Norwalk virus, coxsackievirus,hepatitis A, poliovirus, rhinovirus, severe acute respiratory syndromevirus (SARS), hepatitis C virus, yellow fever virus, dengue virus, WestNile virus, Rubella virus, hepatitis E virus Arenaviridae, Bunyaviridae,Filoviridae, Orthomyxoviridae, Paramyxoviridae and Rhabdoviridae. Theseinclude, but are not limited to influenza virus, Guanarito virus, Juninvirus, Lassa virus, Machupo virus, Sabia virus, Cimean-Congo hemorrhagicfever virus, Ebola virus, Marburg virus, Measelse virus, Mumps virus,Parainfluenza virus Respiratory syncytial virus, human metapneumovirus,Hendra virus, Hipah virus, Rabies virus, Reoviridae including but notlimited to Toravirus, Orbivirus, Coltivirus, Banna virus.Hepatitis D virus.Additional viruses that infect animals include:Rhabdoviridae, including, but not limited to vesicular stomatitis virus.Picornaviridae, including, but not limited to Foot and mouth diseasevirus, Pestiviridae including, but not limited to Classical swine feverand Bovine viral diarrhea.Arteriviridae including, but not limited to equine arteritis virus,porcine reproductive and respiratory syndrome virus, lactatedehydrogenase elevating virus and simian haemorrhagic fever virus.Coronaviridae including, but not limited to infectious bronchitis virus,transmissible gastroenteritis coronoavirus, bovine coronavirus, felinecoronavirus, canine coronavirus, moust hepatitis virus, Toroviridaeincluding, but not limited to Berne virus, Breda virus.Orthomyxoviridae including, but not limited to avian influenza virus,swine influenza virus. Reoviridae including, but not limited toBluetongue virus.Clroviridae including, but not limited to chicken anemia virus, porcinecircovirus-1, porcine cirovirus-2, psittacine beak and feather diseasevirus, pigeon circovirus, canary circovirus and goose circovirus.Asfarviridae including, but not limited to African swin fever virus.Retroviridae including, but not limited to Avian leucosis virus, Roussarcoma virus, mouse mammary tumor virus, murine leukemia virus, felineleukemia virus, boine leukemia virus, Walleye dermal sarcoma virus,Simian and feline immunodeficiency viruses, simian foamy virus.Flaviviridae including, but not limited to Tick-borne encephalitisvirus, Japanese encephalitis virus, St. Louis encephalitis virus, Israelturkey meningoencephalomyelitis virus, Sitiawan virus, Wesselsbron virusand louping ill virus.Paramyxoviridae including, but not limited to canine distemper virus,phocine distemper virus, cetacean morbillivirus, Newcastle diseasevirus, rinder pest virus.

Most Confirmed PS-Interception-Susceptible Enveloped Viruses are RNAViruses:

Ebola and Marburg virus (Filoviridae); Ross River virus, chikungunyavirus, Sindbis virus, eastern equine encephalitis virus (Togaviridae,Alphavirus), vesicular stomatitis virus (Rhabdoviridae, Vesiculovirus),Amapari virus, Pichindé virus, Tacaribe virus, Junin virus, Machupovirus (Arenaviridae, Mammarenavirus), West Nile virus, dengue virus,yellow fever virus (Flaviviridae, Flavivirus); human immunodeficiencyvirus type 1 (Retroviridae, Lentivirus); Moloney murine leukemia virus(Retroviridae, Gammaretrovirus); influenza A virus (Orthomyxoviridae);respiratory syncytial virus (Paramyxoviridae, Pneumovirinae,Pneumovirus)

Confirmed PS-Interception-Susceptible Enveloped DNA Viruses are:

vaccinia virus (Poxviridae, Chordopoxvirinae, Orthopoxvirus); herpessimplex virus type 1, herpes simplex virus type 2 (Herpesviridae,Alphaherpesvirinae, Simplexvirus); human cytomegalovirus (Herpesviridae,Betaherpesvirinae, Cytomegalovirus); Autographa californicanucleopolyhedrovirus (Baculoviridae, Alphabaculoviridae) (an insectvirus)

Prospected PS-Interception-Susceptible Important Enveloped RNA Virusesare:

Ebola and Marburg virus (Filoviridae); Semliki Forest virus, Ross Rivervirus, chikungunya virus, O'nyong-nyong virus, Sindbis virus,eastern/western/Venezuelan equine encephalitis virus (Togaviridae,Alphavirus); rubella (German measles) virus (Togaviridae, Rubivirus);rabies virus, Lagos bat virus, Mokola virus (Rhabdoviridae, Lyssavirus);Amapari virus, Pichindé virus, Tacaribe virus, Junin virus, Machupovirus, Guanarito virus, Sabia virus, Lassa virus (Arenaviridae,Mammarenavirus); West Nile virus, dengue virus, yellow fever virus, Zikavirus, Japanese encephalitis virus, St. Louis encephalitis virus,tick-borne encephalitis virus, Omsk hemorrhagic fever virus, KyasanurForest virus (Flaviviridae, Flavivirus); human hepatitis C virus(Flaviviridae, Hepacivirus); human immunodeficiency virus type 1(Retroviridae, Lentivirus); influenza A/B virus (Orthomyxoviridae, thecommon ‘flu’ virus); respiratory syncytial virus (Paramyxoviridae,Pneumovirinae, Pneumovirus); Hendra virus, Nipah virus (Paramyxoviridae,Paramyxovirinae, Henipavirus); measles virus (Paramyxoviridae,Paramyxovirinae, Morbillivirus)

Prospected PS-Interception-Susceptible Enveloped DNA Viruses are:

variola major (smallpox) virus (Poxviridae, Chordopoxvirinae,Orthopoxvirus); human hepatitis B virus (Hepadnaviridae,Orthohepadnavirus); hepatitis delta virus (hepatitis D virus)(unassigned Family, Deltavirus); herpes simplex virus type 1, herpessimplex virus type 2 (Herpesviridae, Alphaherpesvirinae, Simplexvirus);human cytomegalovirus (Herpesviridae, Betaherpesvirinae,Cytomegalovirus)Anti-PS binding of a molecule to viral envelope PS will thereforeinterfere with acute infection by any of the above named viruses, withacute reactivation of a latent chronic viral infection (in thosePS-labeled viruses capable of maintaining latency), or with chronicviral infection with active viral replication of any of the above namedviruses.The anti-viral actions of PT150, PT155, PT156, PT157, PT158, TCY-1 orany further derivatives therefore may act on any viral infection throughmore than one mechanism of action, either through binding to GREs (withall the possible anti-viral activities noted above) or to PS (with allpossible anti-viral activities noted above) in an additive orsynergistic fashion.

TABLE 5 Anti-viral Activities of PT150 Cytotoxicity SelectivityAntiviral activity (μM) 50% (μM) index 50% Virus Strain Host cell EC₅₀NR Remarks CC₅₀ SI₅₀ Influenza A A/duck/Minne MDCK 13.9 Toxicity on 17.01.2 H5N1 (avian) sota/1525/81 MDCK Rift Valley fever MP-12 Vero 76 12.8Toxicity on 16.5 1.3 Vero 76 Venezuelan equine TC-83 Vero 76 >34.8 Noactivity 34.8 <1 encephalitis Chikungunya S-27 Vero 76 >21.8 No activity21.8 <1 Respiratory A2 (VR-1540) MA-104 >32.5 No activity 32.5 <1syncytial type A Enterovirus D68 US/Kentucky/ RD >10.2 No activity 10.2<1 14-18953 Enterovirus 71 Tainan/4643/98 Vero 76 7.4 Toxicity on 7.41.0 Vero 76 Poliovirus type 3 WM-3 Vero 76 5.6 Toxicity on 7.4 1.3 Vero76 Yellow fever 17D vaccine HeLa 9.8 With hGRα 11.8 1.2 Vero 76 >10.9 NohGRα 10.9 <1 Zika MR766 HuH-7 7.4 Toxicity on 7.4 1.0 HuH-7 Ebola ZaireMayinga Vero >7.9 Toxicity on 7.9 <1 Vero

TABLE 6 Anti-viral Activities of PT155 Cytotoxicity SelectivityAntiviral activity (μM) 50% (μM) index 50% Virus Strain Host cell EC₅₀NR Remarks CC₅₀ SI₅₀ Influenza A A/duck/Minne MDCK 31.2 — >141.7 >4.5H5N1 (avian) sota/1525/81 Rift Valley fever MP-12 Vero 76 10.8 Toxicityon 70.9 6.6 Vero 76 Venezuelan equine TC-83 Vero 76 >18.4 No activity18.4 <1 encephalitis Chikungunya S-27 Vero 76 >12.8 No activity 12.8 <1Respiratory A2 (VR-1540) MA-104 >17.0 No activity 17.0 <1 syncytial typeA Enterovirus D68 US/Kentucky/14- RD >5.8 No activity 5.8 <1 18953Enterovirus 71 Tainan/4643/98 Vero 76 0.51 Toxicity on 45.4 89.0 Vero 76Poliovirus type 3 WM-3 Vero 76 0.74 Toxicity on 45.4 61.4 Vero 76 Yellowfever 17D vaccine HeLa 2.1 With hGRα 6.0 2.9 Vero 76 >6.1 No hGRα 6.1 <1Zika MR766 HuH-7 0.45 Toxicity on 39.7 88.2 HuH-7 Ebola Zaire MayingaVero 7.5 Toxicity on 19.6 2.6 Vero

Influenza Virus

PT150 and PT155 are antivirally active versus influenza A virusreplication at higher concentrations (EC₅₀>>10 μM). PT150 is morepotently active than PT155

This antiviral activity is very probably dependent on humanglucocorticoid receptor-a (hGRa), since PT150 is more active than PT155,with the latter reasonably assumed being a weaker glucocorticoidantagonist than PT150, as was shown for the PT155-related compound PT150oxime (ORG36174). The activity of both compounds is better against avianinfluenza A virus (H5N1) as compared towards swine-origin pandemicinfluenza A (H1N1) virus

The probable mechanism-of-action of PT150 is the trapping of Hsp90 ontoa non-nuclear-translocated complex (PT150-hGRa-Hsp90) hold back in thehost cytosol. Therefore, the amount of free Hsp90 available forinfluenza A virus polymerase complex assembly and nuclear trafficking isdiminished by PT150 and, at lower propensity, also by PT155, leading toantiviral effects.

In addition, inhibition of Hsp90 by small-molecule drugs (for example,geldanamycin/radicicol derivatives) represents an establishedantineoplastic chemotherapeutic strategy. Hsp90 client proteins (>300cellular proteins are known currently to be Hsp90 clients) are importantfor cancer growth and malignant transformation. PT compounds, especiallyPT150, indirectly target Hsp90 by trapping Hsp90 in an inactive complexwith human glucocorticoid receptor isoform alpha. This, in turn, leadsto diminished availability of Hsp90 protein, required for stabilizingclient proteins essential to cancer cell growth, maintenance ofmalignant cell phenotype, tumor angiogenesis, tumor spread andmetastasis.

Rift Valley Fever

PT150 and PT are antivirally active versus Rift Valley fever virusreplication at relatively low concentrations (EC50≈10 μM). PT150 isabout as active as PT155

This antiviral activity is clearly independent of human glucocorticoidreceptor a (hGRa), since Vero 76 cells do not express such a receptor,even not any monkey glucocorticoid receptor. The mechanism-of-action isvery probably inhibition of virus-cell fusion, since RVFV glycoproteinGC contains a binding pocket for lipophilic compounds.

This was substantiated by the observation that the RVFV accessoryproteins (the 78 kDa protein, NSm1, NSm2 and NSs) are dispensable forvirus replication in vitro, and the postulation that the PT compounds donot interact with the large RNA-dependent RNA polymerase L and thenucleocapsid protein N. Other gene expression products are not known,therefore PT compounds should bind to GN and/or GC RVFV envelopetransmembrane glycoproteins mediating receptor binding and fusion

Binding of PT compounds to the nucleocapsid N protein is not expected,since the RVFV N protein does not present hydrophobic binding sites,only a central RNA-binding core (hence, positively charged) andalternately charged N- and C-termini.

A binding pocket for highly lipophilic compounds in the X-raycrystallographic structure of RVFV GC protein was described. GC is thefusion-competent glycoprotein, and represents a class II viral fusionglycoprotein.

Polio Virus

PT150 and PT155 are antivirally active versus poliovirus type 3replication at low concentrations (EC50≈1-5 μM). PT155 is slightly moreactive than PT150

Poliomyelitis is an old disease (see picture). Poliovirus can infectneurons and can lead to neuromuscular paralysis.

The term “poliomyelitis” is used to identify the disease caused by anyof the three serotypes of poliovirus. Two basic patterns of polioinfection are described: a minor illness which does not involve thecentral nervous system (CNS), sometimes called abortive poliomyelitis,and a major illness involving the CNS, which may be paralytic ornonparalytic.

PT150 and PT155 are antivirally active versus poliovirus type 3replication at low concentrations (EC50≈1-5 μM). PT155 is slightly moreactive than PT150

This antiviral activity is clearly independent of human glucocorticoidreceptor-a (hGRa), since Vero 76 cells do not express such a receptor,even not any monkey glucocorticoid receptor. The mechanism-of-action isvery probably inhibition of poliovirus capsid uncoating, since PT150fits well into the picornaviral VP1 capsid protein hydrophobic pocketbinder model like do the “canyon binders” compound 40, arildone,disoxaril, WIN 58084, pleconaril, and pirodavir. For a model of thishydrophobic binding of PT150 into the VP1 pocket of human rhinovirus 14see next side. A similar fit of PT150 is expected into poliovirus type 3VP1 capsid protein hydrophobic pocket.

Hepatitis Virus

ORG34517, PT150, PT155, PT156, PT157, PT158, TCY1 are a new class oftherapeutic agents designed to block the glucocorticoid receptor (GR),acting as an antagonist for endogenous cortisol. The primarydevelopmental pathway to date has been as a treatment forneuropsychiatric diseases characterized by dysregulation of thehypothalamic-pituitary-adrenal axis of signaling that are oftenassociated with higher than normal circulating levels of endogenouscortisol.

Other possible uses include oncology, viral infection and otherneuropsychiatric conditions including post-traumatic stress disorder,weight gain in patients requiring long term anti-psychotic medication,and hospital delirium of the elderly.

Human hepatitis B virus (HBV) and human immunodeficiency virus type 1(HIV-1) integrate their retro-transcribed DNA proviruses into the humanhost genome. Existing antiretroviral drug regimens fail to directlytarget these intrachromosomal xenogenomes, leading to persistence ofviral genetic information. Both HBV and HIV-1 harbor glucocorticoidresponse elements (GREs) in their proviral DNA genomes. This inventiondescribes a potent glucocorticoid antagonist which binds to humanglucocorticoid receptor isoform alpha (hGR-alpha), is translocated tothe nucleus by the nuclear receptor dimer, and has the ability tocovalently inactivate the intragenic and intraexonic viral GREs of HBVand HIV-1. The glucocorticoid antagonist ORG34517 dimericthiosemicarbazone derivative” described by this invention represents thefirst reported antiviral agent capable of eradicating humanimmunodeficiency and hepatitis B proviruses from their human host. Theanti-HBV effect of the glucocorticoid antagonist ORG34517 dimericthiosemicarbazone derivative may be mediated by hGR-alpha, since the HBVgenome contains at least two hGR-alpha trans-activation targets (GREs).The glucocorticoid antagonist ORG34517 dimeric thiosemicarbazonederivative binds to hGR-alpha, is transported through the nuclear porecomplex to the intranuclear HBV genome by the (glucocorticoid antagonistORG34517 dimeric thiosemicarbazone derivative)-liganded hGR-alpha, theliganded hGR-alpha complex binds to GREs, and, finally, theco-transported glucocorticoid antagonist ORG34517 dimericthiosemicarbazone derivative switches to the HBV DNA and covalentlymodifies amine group-containing nucleobases (cytosine, guanine, adenine)by trans-thiocarbamoylation. This could also happen for host cellularGREs, but we think there is selectivity for intraexonic GREs, which arenot typical for the human genome, since viral intraexonic GREs aresubject to different epigenetic regulation involving chromatinremodeling events in comparison to host cellular GREs. Human GREs arelocated in the promoter regions upstream of transcription initiationsites, with only few exceptions where the GRE is found within a humangene intron. Additionally, it should be mentioned that human GREs are inmost instances imperfect (GRE sequence degeneracy), i.e. their sequencesdo not match the perfect GRE consensus sequence given. In spite of theseimperfect GRE sites, their glucocorticoid responsiveness is retained.

GRE 4 is proposed as a target of the glucocorticoid antagonist ORG34517dimeric thiosemicarbazone derivative, since the 5′-LTR GREs are onlypartially functional negative enhancers (silencers) of HIV-1 geneexpression, whereas the GRE 4 in the vif gene was shown to be fullyfunctional as enhancer of HIV gene transcription.

The covalent modification of HIV-1_(LAI) vif gene by the glucocorticoidantagonist ORG34517 dimeric thiosemicarbazone derivative, in closeanalogy to the already described mechanism-of-action on HBV ayw, wouldlead to loss of HIV-1_(LAI) Vif protein (Vif=viral infectivity factor)function. Vif protein serves as an inherent retroviral inhibitor of theinnate human apolipoprotein B mRNA-editing enzyme 3G(APOBEC3G)-dependent human antiretroviral defense system. As aconsequence, Vif could not protect HIV-1 (−)-cDNA towards host APOBEC3Gcytidine deaminase enzymatic activity (dC→dU mutation).

The covalently ‘trapped’ proviral DNA of HBV or HIV-1 is expected toinduce p53-mediated apoptosis through an DNA damage signal mechanismsensored by p53 tumor suppressor protein and/or other DNA damage-inducedsensor mechanisms [e.g. ataxia telangiectasia and Rad3-related protein(ATR)]. By proviral DNA damage-induced apoptosis the host organism ofthe viruses could be successively cleared from host genome-integratedproviral xeno-DNA.

The synthesis of the glucocorticoid antagonist ORG34517 dimericthiosemicarbazone derivative is performed by construction of thethiosemicarbazone of ORG34517 [20 min reflux of ORG34517 with equimolarthiosemicarbazide in 90% (v/v) aqueous ethanol], and its subsequentdimerization by treatment (in acetone, room temperature) with sodiumhydroxide (NaOH, pre-dissolved in water). These transactions are basedon general procedures already described in literature and known to thoseskilled in the art.

The hitherto claimed compounds of this invention could also be activeversus human hepatitis C virus (HCV), which is indicated by a publishedproof-of-concept study. An antiviral mechanism-of-action of theglucocorticoid antagonist ORG34517 dimeric thiosemicarbazone derivative,and of the intermediate ORG34517 thiosemicarbazone, versus humanhepatitis C virus subtype 1b strain Con1 (HCV-1b Con1 is proposed. It isinherently clear that it cannot being mediated by hGR-alpha, since HCVincorporates no DNA stage in its life cycle. Since the anti-HCV-1bactivity of thiosemicarbazones described in this invention wasdetermined with the HCV RNA replicon cell line Huh7 ET (luc-ubi-neo/ET),which only codes for the non-structural HCV proteins NS3, NS4A, NS4B,NS5A and NS5B, the inhibiting action of the thiosemicarbazones describedin this invention must be confined to these five genes and/or geneproducts. It was reported that thiosemicarbazones(5,6-dimethoxyindan-1-one thiosemicarbazone, DMI-TSC) target bovineviral diarrhea virus type 1 (BVDV-1 strain NADL, Flaviviridae,Pestivirus) NS5B protein RNA-dependent RNA polymerase (RdRp). BVDV-1 iscommonly regarded as a suitable surrogate for HCV, because bothFlaviviridae polyprotein sequences are closely related (maximal sequenceidentitiy 39%)

Since the claimed compounds of this invention could also be activeversus Ebola virus Zaire strain 1976 Mayinga (EBOV Zaire 1976 Mayinga),which is indicated by a published proof-of-concept study, a sequencetriple alignment between BVDV-1 NADL NS5B RdRp, HCV-1b Con1 NS5B RdRp,and EBOV Zaire 1976 Mayinga VP40 membrane-associated matrix protein isincluded in this invention. A compound similar to the claimed compoundsof this invention is an inhibitor of EBOV Zaire 1976 Mayingamembrane-associated matrix protein VP40 octamerization, an event whichis essential to EBOV Zaire 1976 Mayinga replication.

The invention provides the treatment of EBOV Zaire infections with theglucocorticoid antagonist ORG34517 dimeric thiosemicarbazone derivative,or the intermediate ORG34517 thiosemicarbazone, both embodiments of thisinvention, as the procedures described in this invention are known tothose skilled in the art.

The invention provides the combination of ORG34517 dimericthiosemicarbazone derivative with additional anti-viral andanti-inflammatory compounds in a kit for the treatment or ameliorationof a condition selected from the group consisting of HBV infection, HIVinfection, and Ebola virus infection.

Cytosolic nuclear receptors translocate into the nucleus followingligand bindingLiganded nuclear receptor dimers bind to hormone response cognateelement DNAGlucocorticoids bind to human glucocorticoid receptor isoformsa/b(hGR-a/b)Liganded hGR-a/bdimer binds to DNA glucocorticoid response elements(GREs)PT155 is metabolically activated by human flavin-containingmonoxygenases (hFMOs) through S-oxidation in the thiosemicarbazone partof PT155The PT155carbodiimide metabolite binds as ligand to the homodimerichGR-a/b, in one half with ORG 34517, and the liganded complextranslocates to the nucleus:hGRsubunit 1-(ORG 34517carbodiimide derivative)=hGRsubunit 2-ORG 34517The hGRa/b-(PT155carbodiimide metabolite) complex binds to GREs inintrachromosomal HBV or HIV-1 proviral DNAThe HBV or HIV-1 host DNA-integrated proviral GRE DNA is therebycovalently inactivated over an amidrazone bondThe covalently trapped proviral DNA of HBV or HIV-1 is expected toinduce p53-mediated apoptos is through an DNA damage signal mechanismsensored by p53 tumor suppressor protein and/or other DNA damage-inducedsensor mechanisms [e.g. ataxiatelangiectasiaandRad3-relatedprotein(ATR)] By proviral DNA damage-induced apoptosis the host organismof the viruses could be successively cleared from host genome-integratedproviral xeno-DNAHuman hepatitis B virus (HBV) and human immunodeficiency virus type1(HIV-1) integrate the irretro-transcribed DNA proviruses into the humanhost genome. Existing antiretroviral drug regimens fail to directlytarget these intrachromosomal xeno genomes, leading to persistence ofviral genetic informationExisting HAART therapies target reverse transcriptase (HBV, HIV),protease (HIV) and integrase (HIV) proteins. These therapy options arenot curativeIn a proof-of-concept study the PT155-similar compound retinazone wasproved to work versus HBV, HIV-1, HCV, and the human herpes virusesHHV-3, HHV-5, HHV-6, and HHV-8 (Herpes viridae)Retinazone's antiviral activity correlated exactly with the presence ofintragenic and intraexonicGREs in virus-essential genes of retinazone-susceptible virusesThe glucocorticoid antagonist ORG34517 thiosemicarbazone derivativePT155 described in this presentation might represent the first reportedantiviral agent capable of eradicating human immunodeficiency andhepatitis B proviruses from their human hostHuman hepatitis C virus (HCV) NS5B RNA polymerase protein bears a commonthiosemicarbazone-binding motifHCV-1b Con1 NS5B fingertip/finger domain RdRp motif I, 139-MAKNEV-144(Hepaciviruscompletely conserved aa residues in bold), was identified asthiosemicarbazone-binding motifIn a proof-of-concept study structurally diverse thiosemicarbazones wereactive as inhibitors of HCV replicationPT155is additionally proposed as HCV inhibitor

HIV The Mechanism-Of-Action of PT150 and PT155 Versus HumanImmunodeficiency Virus Type 1 Strain LAI (Retroviridae, Lentivirus)Results:

PT150 and PT155 are active versus human immunodeficiency virus type 1(HIV-1) strain LAI (HIV-1_(LAI)) in primary human peripheral bloodmononuclear cells (PBMCs) which consist of T lymphocytes, B lymphocytesand monocytes. HIV-1_(LAI) (=HIV-1_(BRu)=LAV-1 was assayed in primary[freshly donated from healthy (tested HIV-1-negative, HBV-negative, andHCV-negative) blood donors, and isolated by single-step Ficoll-Hypaquecentrifugation method] human peripheral blood mononuclear (PBM) cells inthe presence of a drug being evaluated. The parameter for antiviralactivity was reduction of reverse transcriptase (RT) activity in thecell supernatant after Triton X-100-mediated lysis of released virions,as measured by [5alpha-³H]dTTP (5alpha-tritiated thymidine5′-triphosphate) incorporation into poly(rA).poly(dT) directed by theprimed RNA template poly(rA).oligo(dT). It should be noted that theassay did not detect RT inhibition by potential RT inhibitors per se,but indirectly quantified the amount of released HIV-1 in thesupernatant. The detailed assay methodology was reported by Schinazi etal., as based on an older assay system of Spira et al. The experimentswere conducted in triplicate and treated statistically by regressioncurve analysis (r² coefficient of determination). The RT inhibitor AZT(zidovudine, 3′-azido-3′-deoxythymidine; RETROVIR™) served as a positivecontrol. Cytotoxicity on PBMC exerted by the test compounds wasdetermined as described by Stuyver et al., by application of theCellTiter 96® AQ_(ueous) One Solution Cell Proliferation Assay (PromegaCorp., Madison, Wis.). Briefly, the phenazine ethosulfate (PES)-coupledreduction of the tetrazolium salt3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) to a purple formazan by undamaged cells was measured.

Mechanism-of-Action:

The antiretroviral action of PT150 and PT155 is mediated via DNAglucocorticoid response elements (GREs) residing in the prolentivirus ofHIV-1_(LAI) integrated into the nuclear, chromosomal human host genomeas xenogenomic prolentiviral DNA sequence: Cytosolic nuclear receptorstranslocate into the nucleus following ligand binding Liganded nuclearreceptor dimers bind to hormone response cognate element DNAGlucocorticoids bind to human glucocorticoid receptor isoform alpha(hGR-alpha) Liganded hGRalpha dimer binds to DNA glucocorticoid responseelements (GREs) Glucocorticosteroid nuclear receptors target GRE DNA

HIV-1_(LAI) provirus (9229 nt) harbors four GREs: GRE 1-4HIV-1_(L)m vif intragenic GRE 4 is located in vif gene exonHIV-1_(LAI) proviral DNA contains 4 recognized GREs (GRE 1, GRE 2, GRE3, GRE 4)GRE 1 (-264 to −259), GRE 2 (-6 to −1) and GRE 3 (+15 to +20) areLocated within the 5′-Long Terminal Repeat (5′-LTR)PT155 is metabolically activated by human flavin-containingmonoxygenases (hFMOs) through 5-oxidation in the thiosemicarbazone partof PT155.The PT155 carbodiimide metabolite binds as ligand to the homodimerichGRalpha, in one half with PT150, and the liganded complex translocatesto the nucleus:

hGRalpha subunit 1-(PT155 carbodiimide metabolite)≡hGRalphasubunit2-PT150 The hGRalpha-(PT155 carbodiimide metabolite) complex binds toGREs in intrachromosomal HIV-1 proviral DNA

Consequences of Proviral DNA Trapping by the PT155 CarbodiimideDerivative:

The covalently trapped proviral DNA of HIV-1 is expected to inducep53-mediated apoptosis through an DNA damage signal mechanism sensoredby p53 tumor suppressor protein and/or other DNA damage-induced sensormechanisms [e.g. ataxia telangiectasia and Rad3-related protein (ATR)]

By proviral DNA damage-induced apoptosis the host organism of theviruses could be successively cleared from host genome-integratedproviral xeno-DNA

Without being bound by any theory, it is believed that Humanimmunodeficiency viruses (HIVs) integrate their retro-transcribed DNAproviruses into the human host genome. Existing antiretroviral drugregimens fail to directly target these intrachromosomal xenogenomes,leading to persistence of viral genetic information. Existing HAARTtherapies target reverse transcriptase, protease and integrase proteins.These therapy options are not curative. In a proof-of-concept study thePT155-similar compound retinazone was proved to work versus HBV, HIV-1,HCV, and the human herpesviruses HHV-3, HHV-5, HHV-6, and HHV-8(Herpesviridae). Retinazone's antiviral activity correlated exactly withthe presence of intragenic and intraexonic GREs in virus-essential genesof retinazone-susceptible viruses. The glucocorticoid antagonist PT155described in this presentation might represent the first reportedantiviral agent capable of eradicating human immunodeficiency provirusesfrom their human host

The Metabolic Activation of PT150 Thiosemicarbazone Contained in PT155:S-Oxidation by Human Flavin-Containing Monooxygenases (hFMOs):

It is well-known that thiosemicarbazones like thiacetazone(p-acetamidobenzaldehyde thiosemicarbazone), a cheap, second-lineantitubercular substance discovered by Nobel laureate Gerhard Domagk in1946, are bio-activated by human flavin-containing monooxygenases(hFMO1, hFMO2.1, hFMO3) into a sulfenic acid (R—S—OH), a sulfinic acid[R—(S═O)—OH], and a carbodiimide derivative (R—N═C═N—H). Both thesulfenic acid and the carbodiimide metabolite are the activeantitubercular metabolites. The sulfinic acid does not form thecarbodiimide, and represents an inactive metabolite. Especially thecarbodiimide metabolite can react with amino acid residues in targetproteins of, for example, Mycobacterium tuberculosis, or with humanhepatic/extrahepatic glutathione R—SH thiol group for metabolicdetoxification: Therefore, PT155 will be bio-activated (S-oxidized) inthe same manner as thiacetazone by hFMOs under consumption of molecularoxygen (O₂), and will be able to bind covalently to HBV and HIV proviralGRE DNA after being shuttled to the host cell nucleus by hGRalpha.

Human flavin-dependent monooxygenases (hFMOs) (EC 1.14.13.8) are thesecond important type of monooxygenases in the human body, the otherwell-known type being the cytochrome P450 monooxygenases (CYP450monooxygenases). Both are localized in microsomes and dependent onmolecular oxygen (O₂), and hFMOs need also the cofactors flavin-adeninedinucleotide (FAD) and nicotinamide-adenine dinucleotide phosphate(NADPH), whereas CYP450 monooxygenases are heme-dependent. hFMO1 ismainly expressed in human kidney, and, to a smaller extent, in smallintestine and lung. hFMO2 is expressed to a very high level in humanlung and kidney, and, to a smaller extent, in liver and small intestine.hFMO3 and hFMO5 are highly expressed in human liver, but are alsoexpressed in lung and, to a smaller extent, in human kidney. hFMO4 ismainly expressed in kidney and, to a smaller extent, in liver and smallintestine. hFMO5 is also expressed to a high level in small intestine.hFMO2 is additionally expressed in human brain, but at low abundance(<1% of lung).

Slightly Modified Citation Following:

“The hFMOs oxygenate nucleophilic heteroatom-containing chemicals anddrugs and generally converts them into harmless, polar, readily excretedmetabolites. Sometimes, however, FMO bioactivates chemicals intoreactive materials that can cause toxicity. Most of the interindividualdifferences of hFMOs are due to genetic variability and allelicvariation, and splicing variants may contribute to interindividual andinterethnic variability observed for hFMO-mediated metabolism. Incontrast to cytochrome P450 monooxygenases (CYP450 monooxygenases),hFMOs are not easily induced nor readily inhibited, and potentialadverse drug-drug interactions are minimized for drugs prominentlymetabolized by hFMOs. These properties may provide advantages in drugdesign and discovery, and by incorporating hFMOdetoxication pathwaysinto drug candidates, more drug-like materials may be forthcoming.Although exhaustive examples are not available, physiological factorscan influence hFMOfunction, and this may have implications for theclinical significance of hFMOs and a role in human disease.”

Implications of the Structure of PT155 on Nuclear ReceptorBinding—Nuclear Translocation as Key to Biological Action of PT155:

PT150 does not support nuclear translocation of the PT150-hGRalphacomplex. It is a competitive antagonist to cortisol in the cytosol only.Therefore, to reach our goal to target HBV and HIV proviruses residingin eukaryotic cell nuclear chromatin we must force nucleartranslocation. Nuclear translocation of glucocorticosteroid-hGRcomplexes relies on structural modalities. The RU38486 (RU486,mifepristone) RU486-hGRalpha complex is indeed translocated into thenucleus, since RU486 represents a partial agonist at hGRalpha. Theresidual agonist potency of RU486 is sufficient to induce nucleartranslocation. This latter property can be viewed as being strictlydependent of hGRalpha protein conformation with bound RU486 which stillenables steroid receptor coactivator 2 (SRC-2) [=nuclear receptorcoactivator 2 (NCoA-2),=glucocorticoid receptor interacting protein 2(GRIP2),=transcriptional intermediary factor 2 (TIF-2)] or SRC-1 bindingas necessary condition for nuclear targeting. These conformationalinductions of RU486 on hGRalpha protein structure are pictured [nextpage, top and bottom].

From these Considerations of the Three-Dimensional hGRalpha ProteinStructure it can be Deduced that the Dimer Postulated Before is Indeednot Able to Bind to hGRalpha Because its Size is Too Large to Fit in thehGRalpha Ligand-Binding Domain (LBD) Pocket. It Certainly would notInduce Nuclear Translocation.

TABLE 7 Antiviral Activity of PT155 against Inducible HIV-1LAV inOM-10.1 Cells Selectivity Index Test Article EC₅₀ (μM) EC₅₀ (μM) CC₅₀(μM) (CC₅₀/EC₅₀) PT155 49.4 4.33 >100 >23.1 Temacrazine 0.640.07 >1.00 >0.13 (TMZ)

TABLE 8 Inhibition of HIV-1 LAV in OM-10.1 by TMA RT Values (cpm) CONC(nM) 0.0 0.10 0.32 1.00 3.16 10.0 31.6 100 316 1000 SAMPLE 1 28674 3038028496 32770 30588 27453 25515 18782 6512 1016 SAMPLE 2 29640 31745 3101323041 25259 31623 24677 9733 5272 471 SAMPLE 3 33105 26409 29181 2450428648 31990 29321 5790 8986 351 MEAN 30472.8 29511.5 29563.5 26771.828165.2 30355.5 26504.5 11435.2 6923.5 312.8 % VC 100.0 96.8 97.0 87.992.4 99.6 87.0 37.5 22.7 2.0 STD DEV 7.6 9.1 4.3 17.2 8.9 8.3 8.1 21.96.2 1.2 TOXICITY VALUES (Cell Titer 96-O.D. @ 490/650 nm) CONC (nM) 0.00.10 0.32 1.00 3.16 10.0 31.6 100 316 1000 SAMPLE 1 0.740 0.727 0.7290.781 0.830 0.882 0.967 0.917 0.977 0.897 SAMPLE 2 0.715 0.895 0.8270.744 0.933 0.999 0.902 0.990 1.037 0.910 SAMPLE 3 0.614 0.621 0.7680.736 0.849 0.954 0.988 0.952 0.813 0.879 MEAN 0.690 0.748 0.775 0.7540.871 0.945 0.952 0.953 0.942 0.896 % CC 100.0 108.4 112.4 109.3 126.3137.0 138.1 138.2 136.7 129.9 STD DEV 9.7 20.0 7.1 3.5 8.0 8.6 6.5 5.316.8 2.2 DRUG: TMZ 50% 90% 95% TC (nM) >1000 >1000 >1000 IC (nM) 74.8641 847 ANTIVIRAL INDEX >13.4 >1.56 >1.18 (AI)

TABLE 9 Inhibition of HIV-1 LAV Replication in OM-10.1 by PT155 RTValues (cpm) CONC (μM) 0.0 0.01 0.03 0.10 0.32 1.00 3.16 10.0 31.6 100SAMPLE 1 28674 32320 33018 29801 29905 23474 16156 8592 4254 0 SAMPLE 229640 29982 33056 29663 25409 26693 21122 7827 4766 247 SAMPLE 3 3310529144 32387 31726 34136 24955 15662 10011 5466 454 MEAN 30472.8 30482.232820.5 30396.8 29816.8 25040.8 17646.8 8810.2 4828.5 233.8 % VC 100.0100.0 107.7 99.8 97.8 82.2 57.9 28.9 15.8 0.8 STD DEV 7.6 5.4 1.2 3.814.3 5.3 9.9 3.6 2.0 0.7 TOXICITY VALUES (Cell Titer 96-O.D. @ 490/650nm) CONC (μM) 0.0 0.01 0.03 0.10 0.32 1.00 3.16 10.0 31.6 100 SAMPLE 10.740 0.707 0.749 0.867 0.925 0.937 0.896 0.828 0.714 0.777 SAMPLE 20.715 0.669 0.794 0.820 0.933 0.911 0.800 0.761 0.682 0.775 SAMPLE 30.614 0.567 0.642 0.590 0.635 0.618 0.572 0.599 0.536 0.775 MEAN 0.6900.648 0.728 0.759 0.831 0.822 0.756 0.729 0.644 0.776 % CC 10.0 93.9105.6 110.0 120.5 119.2 109.6 105.7 93.4 112.5 STD DEV 9.7 10.5 11.421.5 24.7 25.7 24.2 17.1 13.8 0.1 DRUG: PT-155 50% 90% 95% TC(μM) >100 >100 >100 IC (μM) 4.33 49.4 72.4 ANTIVIRALINDEX >23.1 >2.02 >1.38 (AI)

Zika and Flavivirus

The Flavivirus family (Flaviviridae) are single-stranded (+) RNA virus.Other pathogens of note in the Flavaviridae include Hepatitis C, YellowFever, West Nile, St. Louis Encephalitis, Japanese Encephalitis andDengue Fever. They are vector-transmitted (mosquito or tick) andgenerally causes mild, flu-like infections lasting less than a week. TheFlavivirus have a unique set of secondary structures in their 3′untranslated regions (UTRs) which play roles in their replication aswell as in their ability to produce subgenomic flavivirus (sfRNAs) whendigested by host exonucleases. sfRNAs are of particular interest, asthey are believed to play roles in altering host metabolic pathwaysthrough alteration of host mRNA stability, RNAi and DICER activity(Roby, 2014). sfRNAs have been implicated in altering the immuneresponse to promote viral pathogenicity (Chang et al., 2013) as well asa recently discovered potential link between Zika, microencephaly andGuillain-Barré Syndrome (Ricketson & Lyons-Weiller, 2016).

Zika originated in central western African and spread through parts ofAfrican, Asia and Micronesia through the early part of the 21^(st)century. Introduction into the Americas raised concern when the virusbegan to spread rapidly in Brazil (2015). The large number of cases haveco-occurring have demonstrated the additional dangers of Zika causingmicrocephaly and other birth defects in the fetuses of pregnant women(Johannson et al., 2016; Malkki, 2016) and in the increased incidence ofGuillain-Barré Syndrome (Cao-Lormeau et al., 2016; Paploski, 2016).

Microcephaly results from both genetic and environmental factors.Genetic factors are estimated to occur in 1:30-50,000 live births, whileenvironmental factors are more common, estimated to occur in 1:10,000live births. The connection between ZIka and microencephaly was made inBrazil in 2015 (Moron et al., 2016; Saiz et al., 2016; Slavov et al.,2016), and although the data is preliminary and difficult to correlate,it appears that case of microcephaly rose in infected areas of Brazil asmuch as 4-5 times in the northeast states of that country in 2015(Butler, 2016). The primary risk appears to coincide with infectionduring the first trimester of pregnancy; Cauchenez et al. (2016) modeleddata from cases in French Polynesia and estimated an increased risk formicrocephaly 47.5 times greater in the first trimester if Zika infectionoccurred.

The CDC reported 3,988,076 births in the United States in 2014 (CDCVital Statistics Reports, 2015). Births per month remain fairly steadyin the United States with the highest months being July and August eachyear (Live Science, 2010). Extrapolating from 2006 data, 56.64% ofchildren in 2016 will be conceived between the months of March throughSeptember with Zika-infected mosquitoes spreading throughout thecountry. That would place a 2,258,846 births in the upcoming year atincreased risk for microcephaly. Using a 1:10,000 ratio as a guide, thatwould mean a possible increase of microcephaly in the Unites States inthe upcoming year of as much as 9.3%.

Guillain-Barré syndrome (GBS) is an autoimmune disorder of theperipheral nervous system. The causes of GBS are elusive but have beendemonstrated to occur following bacterial of viral infections. It hasbeen theorized that inappropriate stimulation of the immune system bysome infections is key to the induction of GBS. GBS is treated by plasmaexchange or immunoglobulin therapy to lessen its duration and severity.Glucocorticoid therapy, useful in other autoimmune conditions, has beendemonstrated to increase the severity of GBS.

To date, only one research study (Cao-Lormeau et al., 2016) hasdemonstrated Zika Virus as the causative agent of GBS. Yung & Thoon(2016) have modeled the increased risk of GBS following Zika infectionusing data gathered from French Polynesia in 2015. They report inincreased incidence of GBS from a baseline of 0.24 per 1000 to 0.41 per1000. This equates to a 21-fold risk increase of GBS.

Using 2004 data from the CDC, Frenzen (2008) calculated the annualhealth care cost for GBS in the United States at $1.7B dollars annually.CDC 2015 data lists the frequency of GBS in the United States atapproximately 1:100,000. The mortality of GBS has been estimated inseveral studies Alshekhlee et al. (2008) at 2.58% and at 3.9% (van denBerg, 2013) but can vary widely because of demographics such as age andaccess to health care. Extrapolation from these statistics presents avery chilling scenario, where a fast-spreading population of Zikainfection can cause a cascade of reactions impacting the number of GBScases, health care costs and mortality rates.

The adverse reaction of patients receiving glucocorticoids with GBS isof particular interest with the compounds of the invention, as theydemonstrate not only in vitro antiviral activity but also glucocorticoidantagonism. This means the compound PT150, which has already beenestablished as safe in Phase II Human Clinical Trials, and the compoundPT155, are potentially able to treat not only the infection but decreasethe risks of contracting GBS and/or mitigating its severity as apost-infection occurrence. PT150 (formerly known as ORG34517) hasactivity versus Flaviviridae, including Zika virus and yellow fevervirus, and, possibly, some Picornaviridae like poliovirus. The proposedmechanism-of-action is as follows: PT150 represents a known, andclinically validated, potent and selective glucocorticoid receptorantagonist. Most Flaviviridae (excluding the genus Flavivirus) and allPicornaviridae contain an internal ribosome entry site (IRES) at the5′-end of their (+)-ssRNA genome, a 5′-noncoding region (5′-NCR). ThisIRES is in turn necessary for translation at human host cell ribosomesof the viral (+)-ssRNA genome into a polyprotein (which is cleaved byhost cellular and virally-encoded proteases to yield mature viralproteins)

However, there is also a highly conserved noncoding region (NCR) at the3′-end of the (+)-ssRNA genome, the 3′-NCR which exhibits a highlyconserved stem-loop-containing RNA secondary structure (refs 22, 23)(see graphic next side)

The 3′-NCR binds several host cell proteins, one being hVIP/Mov34 whichis required for proper Flavivirus propagation(transcription/replication). hVIP/Mov34 (human Vpr-interacting protein)is a member of the eukaryotic initiation factor 3A (eIF3) family (ref.26), and is required for Flaviviridae 3′-NCR-controlled replication (theexact roles of the 3′-NCR in Flaviviridae replication are presentlyunknown and remain to be elucidated). This was specifically proved forthe 3′-NCR of Japanese encephalitis virus (JEV), a typical Flaviviruslike Zika virus.

A Mov34-homologous protein serves as a 26S proteasome S12 subunit p40(the proteasome is the “waste container” of the human cell and degradesall overused, misfolded and “trash” proteins by protease digestionwithin an enormously large protein complex, called the 26S proteasome)Mov34-like proteins are an integral part of eukaryotic initiation factor3 (eIF3) complex: eIF3 subunit F p47 (37.5 kDa) & eIF3 subunit H p40(39.9 kDa). Therefore, Mov34-like proteins seem to be life-essential andmulti-functional HIV-1 vpr accessory gene product protein Vpr interactswith the human Mov34 protein, therefore Mov34 was termed: humanVpr-interacting protein (hVIP/Mov34). Mov34 was linked to the G2/M phasetransition of the mammalian cell cycle, that means that hVIP/Mov34protein is essential for the transition from G2 to M phase of human celldivision. The carboxyl terminus of hVIP/Mov34 is critical for HIV-1-Vprinteraction and glucocorticoid-mediated signaling. This defines thecrucial interaction between human Mov34 protein and human glucocorticoidreceptor alpha.

In the absence of Vpr or HIV-1 infection, full-length hVIP/Mov34 isexpressed in the cytoplasm. The cytoplasmic localization pattern offull-length hVIP/Mov34 protein, however, is shifted to a clear nuclearlocalization pattern in cells expressing both hVIP/Mov34 and HIV-1 Vpr.In contrast, Vpr did not alter the localization pattern of hVIP/Mov34mutants, which have their carboxyl-terminal domain deleted. The movementof hVIP/Mov34 supported prior work that suggested that Vpr triggersactivation of the glucocorticoid receptor complex. It was observed thatdexamethasone moves hVIP/Mov34 into the nucleus and that mifepristone(RU38486) inhibited this effect. Interestingly, the expression of anhVIP/Mov34 carboxyl-terminal mutant, which is not responsive to Vpr, isalso not responsive to dexamethasone. These data illustrate that thecarboxyl-terminal domain of hVIP/Mov34 is critical for mediatinghVIP/Mov34-Vpr interaction as well as for its hGRα response. Theseresults support the view that hVIP/Mov34 is a member of the complexarray of nucleocytoplasmic shuttling proteins that are regulated byHIV-1 infection and hGRalpha. Mov34 protein from mouse brain interactswith the 3′-noncoding region (3′-NCR) of Japanese encephalitis virus.Therefore, as Mov34 is a hGRa-binding partner, the 3′-NCR offlaviviruses is human glucocorticoid receptor-regulated and, therefore,prone to inhibition by glucocorticoid antagonists. Flaviviruses needMov34 to replicate optimally in human cells. The glucocorticoidantagonist mifepristone (RU38486) was shown to inhibit Mov34 function inthe case of HIV-1 infection. PT150 and, presumably, also PT155 bind asantagonists to human glucocorticoid receptor isoform alpha (hGRalpha).Human glucocorticoid receptor isoform b (hGRb) is an inactive receptor.PT150 and/or PT155-liganded hGRa, also Hsp90-bound inactive complex, inturn sequesters human Mov34 protein. This leads to a block of Flavivirusreplication, since Mov34 is made unavailable for binding to yellow feverand Zika virus 3′-noncoding region (3′-NCR). PT150 and PT155 act viahVIP/Mov34 protein on Flavivirus 3′-noncoding region, which is also theorigin of subgenomic flavivirus RNA (sfRNA) important for pathogenicityand immune evasion. hVIP/Mov34 protein is human glucocorticoid receptora-regulated, and binds both to hGRα and HIV-1 Vpr protein. hVIP/Mov34protein is indispensable for optimal replication and host pathogenicityof flaviviruses, this effect requires binding of hVIP/Mov34 to 3′-NCRand to the 3′-NCR-derived sfRNA hVIP/Mov34 protein binds to Flavivirus3′-NCR and sfRNA, the exact binding mode and further details are stillunknown

PT155 is more potent than PT150 regarding inhibition of Flavivirusreplication, as is PT155 versus HIV-1 replication.The Filoviridae are a Family within the Order MononegaviralesThe Filoviridae consist of (in 2014):Genus Marburgvirus: Marburg virus, Ravn virusGenus Ebolavirus: Taï Forest virus, Reston virus, Sudan virus, Ebolavirus, Bundibugyo virus Genus Cuevavirus: Lloviu virusFiloviridae are enveloped, non-segmented negative sense single-strandedRNA viruses [(−)-ssRNA viruses]Filoviridae exhibit similarities to the Rhabdoviridae, Paramyxoviridaeand Bornaviridae. Together they build up the Order Mononegavirales.The compounds retinazone (RTZ) and PT155 share structural elements(lipophilic core/thiosemicarbazone head) required for the inhibition ofEBOV matrix protein VP40 octamerization and ring-mediated RNA bindingRTZ was already proved to act as an in vitro inhibitor of EBOVreplication in Vero cells.RTZ inhibits EBOV matrix protein VP40 octamerizationWe show here now that PT155 also acts as an in vitro inhibitor of EBOVreplication, and, analogously to RTZ, probably acts by inhibition ofEBOV matrix protein VP40 octamerization andRNA binding in Vero cells (first antifiloviral mechanism-of-action) Inaddition, PT150 and PT155 also antagonize Hsp90 in cells expressing hGRα(second antifiloviral mechanism-of-action).

Results:

The compound PT155 is highly active versus the very first isolate ofZika virus (MR766) obtained from rhesus monkey 766 at Zika Forest(Uganda) in 1947. This activity is seen in HuH-7 human hepatocellularcarcinoma (HCC, hepatoma) cells, established from a liver tumor in a57-year-old Japanese male in 1982 by Nakabayashi & Sato). In cells notexpressing functional human glucocorticoid receptor-alpha (hGRalpha)PT155 is generally not active versus Zika virus MR766. This stronglypoints to involvement of hGRalpha in the antiviral activity of PT155versus Zika virus MR766.

Putative Mechanism-of-Action:

Most Flaviviridae (excluding the genus Flavivirus) and allPicornaviridae contain an internal ribosome entry site (IRES) at the5′-end of their (+)-ssRNA genome, a 5′-noncoding region (5′-NCR). ThisIRES is in turn necessary for translation at human host cell ribosomesof the viral (+)-ssRNA genome into a polyprotein (which is cleaved byhost cellular and virally-encoded proteases to yield mature viralproteins). However, there is also a highly conserved noncoding region(NCR) at the 3′-end of the (+)-ssRNA genome, the 3′-NCR which exhibits ahighly conserved stem-loop-containing RNA secondary structure.

The 3′-NCR binds several host cell proteins, one being hVIP/Mov34 whichis required for proper Flavivirus propagation(transcription/replication). hVIP/Mov34 (human Vpr-interacting protein)is a member of the eukaryotic initiation factor 3A (eIF3) family, and isrequired for Flaviviridae 3′-NCR-controlled replication (the exact rolesof the 3′-NCR in Flaviviridae replication are presently unknown andremain to be elucidated). This was specifically proved for the 3′-NCR ofJapanese encephalitis virus (JEV), a typical Flavivirus like Zika virus.Glucocorticoid receptor (GR) agonists (dexamethasone) and glucocorticoidantagonists (mifepristone, RU38486) interact with hVIP/Mov34.Specifically, GR agonists stimulate hVIP/Mov34 translocation into thenucleus, and mifepristone inhibits this translocation. Therefore,mifepristone can act as an hVIP/Mov34 antagonist, and this antagonism isable to suppress Flaviviridae replication, as was disclosed in an U.S.Pat. Appl. for the Hepacivirus human hepatitis C virus, by yet unknowndetailed mechanism(s).

Vero cell derivatives (Vero, Vero 76, Vero E6) do not contain humanglucocorticoid receptor, as they are derived from African green monkey(Chlorocebus aethiops, Cercopithecus aethiops) epithelial kidney cells.They even do not contain a Chlorocebus glucocorticoid receptor, and Verocells do not respond to dexamethasone. Therefore, in all Vero cellderivatives (Vero, Vero 76, Vero E6) an antiviral effect of PT155 versusFlaviviridae will be obscured.

hVIP/Mov34 protein may be crucial to Flavivirus replication, infectivityand pathogenicity, and key to the antiflaviviral activity of PTcompounds. Mov34 protein is named correctly: Moloney murine leukemiaprovirus insertion-disrupted protein of 36 kDa:Mouse (murine) Mov34 gene was firstly identified in 1987 during mutationanalysis of Moloney murine leukemia virus (MoMLV) provirus insertionmapping in mice: Retroviruses and insertional mutagenesis in mice:proviral integration at the Mov 34 locus leads to early embryonic deathMouse (murine)Mov34 gene was further investigated in 1990, includingchromosome mapping of a human Mov34 homolog gene: Molecular analysis ofthe Mov 34 mutation: transcript disrupted by proviral integration inmice is conserved in Drosophila. Mouse (murine)Mov34 gene was cloned andsequenced in 1991 Mouse (murine) Mov34 protein expression is essentialto embryonic development: Embryos homozygous at the Mov34 locus developnormally to the blastocyst stage and die shortly after implantation,indicating that virus integration resulted in a recessive lethalmutation. A Mov34-homologous protein serves as a 26S proteasome S12subunit p40 (the proteasome is the “waste container” of the human celland degrades all overused, misfolded and “trash” proteins by proteasedigestion within an enormously large protein complex, called the 26Sproteasome) Mov34-like proteins are an integral part of eukaryoticinitiation factor 3 (eIF3) complex: eIF3 subunit F p47 (37.5 kDa) & eIF3subunit H p40 (39.9 kDa)Therefore, Mov34-like proteins seem to be life-essential andmulti-functional HIV-1 vpr accessory gene product protein Vpr interactswith the human Mov34 protein, therefore Mov34 was termed: humanVpr-interacting protein (hVIP/Mov34). Mov34 was linked to the G₂/M phasetransition of the mammalian cell cycle, that means that hVIP/Mov34protein is essential for the transition from G₂ to M phase of human celldivision.

The carboxyl terminus of hVIP/Mov34 is critical for HIV-1-Vprinteraction and glucocorticoid-mediated signaling. This defines thecrucial interaction between human Mov34 protein and hGRalpha.

In the absence of Vpr or HIV-1 infection, full-length hVIP/Mov34 isexpressed in the cytoplasm. The cytoplasmic localization pattern offull-length hVIP/Mov34 protein, however, is shifted to a clear nuclearlocalization pattern in cells expressing both hVIP/Mov34 and HIV-1 Vpr.In contrast, Vpr did not alter the localization pattern of hVIP/Mov34mutants, which have their carboxyl-terminal domain deleted. The movementof hVIP/Mov34 supported prior work that suggested that Vpr triggersactivation of the glucocorticoid receptor complex. It was observed thatdexamethasone moves hVIP/Mov34 into the nucleus and that mifepristone(RU38486) inhibited this effect. Interestingly, the expression of anhVIP/Mov34 carboxyl-terminal mutant, which is not responsive to Vpr, isalso not responsive to dexamethasone. These data illustrate that thecarboxyl-terminal domain of hVIP/Mov34 is critical for mediatinghVIP/Mov34-Vpr interaction as well as for its hGRalpha response. Theseresults support the view that hVIP/Mov34 is a member of the complexarray of nucleocytoplasmic shuttling proteins that are regulated byHIV-1 infection and hGRalpha. Mov34 protein from mouse brain interactswith the 3′-noncoding region (3′-NCR) of Japanese encephalitis virus.Therefore, as Mov34 is a hGRalpha-binding partner, the 3′-NCR offlaviviruses is human glucocorticoid receptor-regulated and, therefore,prone to inhibition by glucocorticoid antagonists. Flaviviruses needMov34 to replicate optimally in human cells. The glucocorticoidantagonist mifepristone (RU38486) was shown to inhibit Mov34 function inthe case of HIV-1 infection. PT155 binds as antagonist to humanglucocorticoid receptor isoform alpha (hGRalpha) [human glucocorticoidreceptor isoform alpha (hGRalpha) is an inactive receptor beingligand-independent]. PT155-liganded hGRalpha, also Hsp90-bound inactivecomplex, in turn sequesters human Mov34 protein. This leads to a blockof Flavivirus replication, since Mov34 is made unavailable for bindingto yellow fever and Zika virus 3′-noncoding region (3′-NCR).

hVIP/Mov34 Protein Binds to Highly Secondary-Structured Stem-Loop RNA ofFlavivirus 3′-NCR:hVIP/Mov34 Protein Interacts with the 3′-Noncoding Region(3′-NCR)-Derived Subgenomic Flavivirus RNA (sfRNA) which is Generated byHuman Host Cell XRN1 Exoribonuclease (5′→3-exoRNase)

Summary of the Mechanism-of-Action of PT155 Versus the FlavivirusCongeners Yellow Fever Virus and Zika Virus:

PT155 acts via hVIP/Mov34 protein on Flavivirus 3′-noncoding region,which is also the origin of subgenomic flavivirus RNA (sfRNA) importantfor pathogenicity and immune evasion hVIP/Mov34 protein is hGR

gulated, and binds both to hGRalpha and HIV-1 Vpr protein hVIP/Mov34protein is indispensable for optimal replication and host pathogenicityof flaviviruses, this effect requires binding of hVIP/Mov34 to 3′-NCRand to the 3′-NCR-derived sfRNA hVIP/Mov34 protein binds to Flavivirus3′-NCR and sfRNA, the exact binding mode and further details are stillunknownPT155 is more potent than PT150 regarding inhibition of Flavivirusreplication, as is PT155 versus HIV-1 replicationActivity of PT150 and PT155 versus Flavivirus replication can only bedemonstrated in cells expressing human glucocorticoid receptor-alphaSearch for a hVIP/Mov34 Cofactor:

As HIV-1 Vpr protein acts as a coactivator for hGRalpha and is dependenton hVIP/Mov34 to exert this coactivator function, it is highly probablethat hVIP/Mov34 analogously needs a cofactor for binding to Zika virus3′-NCR in conjunction with hGRalpha. Since it was found that PTcompounds, including PT155, are inactive versus dengue virus type 2strain New Guinea C (NGC) replication in hGRalpha-expressing HuH-7cells, a Basic Local Alignment Search Tool (BLAST®) BLASTP 2.3.1+ searchwas conducted for the viral protein product of maximal sequencedifference between Zika virus MR766 and dengue virus type 2 NGC. Thiswas anticipated to yield the hVIP/Mov34 cofactor required for hGRalpharecruitment to Zika virus 3′-NCR, since this putative Zika viruscofactor was expected to be dysfunctional in dengue virus type 2. Inturn, this would explain the inactivity of PT compounds, includingPT155, versus dengue virus type 2.

It was found that the flaviviral non-structural protein 2A (NS2A)exhibited least similarity, or evolutionary conservation, between Zikavirus and dengue virus type 2. The absolute amino acid identity was only29%, whereas overall polyprotein absolute amino acid identity was 56%.This clearly pointed to involvement of Zika virus NS2A protein inantiviral action of PT155. The inactivity of PT compounds versus denguevirus type 2 is caused by missing affinity of PT compounds to thedivergent dengue virus type 2 NS2A protein.

This coincidence was proved by a report that found strong physicalbinding of flaviviral NS2A protein to the corresponding Flavivirus3′-NCR. In a comprehensive recent tabulation of all viral and hostcellular factors known to bind to Flavivirus 3′-NCR, both hVIP/Mov34 andNS2A are indicated as high-affinity ligands for flaviviral 3′-NCR

The Mechanism-Of-Action of PT155 and PT156 Versus Human Hepatitis CVirus (Flaviviridae, Hepacivirus) HuH-7 Clone B (Genotype 1a) RepliconResults:

PT155 and PT156 are active within human hepatitis C virus (HCV) genotype1a (HCV-1a) HuH-7 cell-derived subgenomic replicon established by C. MRice & colleagues in 2000. The sequence of this replicon is based on theHCV-1a (isolate H77) genome. PT156 is more active than PT155, and PT150shows only marginal activity versus HCV-1a (isolate H77) HuH-7cell-derived subgenomic replicon RNA replication.

Mechanism-of-Action:

The antihepaciviral action of PT155 and PT156 is mediated by thethiosemicarbazone motif, or the 4-phenylthiosemicarbazone motif,respectively:

Human hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase (RdRp)protein bears a common thiosemicarbazone-binding motif.HCV-1a (isolate H77) NS5B fingertip/finger domain RdRp motif I,represents a thiosemicarbazone-binding motif.

5,6-Dimethoxyindan-1-one thiosemicarbazone (DMI-TSC) was reported toinhibit bovine viral diarrhea virus type 1 (BVDV-1 strain NADL,Flaviviridae, Pestivirus) NS5B RdRp.

In a proof-of-concept study structurally diverse thiosemicarbazones werefound to be active as inhibitors of HCV genotype 1b (HCV-1b) (isolateCon1) replicon RNA replication.The antihepaciviral pharmacophore in PT155 and PT156 is thethiosemicarbazone moiety, or the 4-phenylthiosemicarbazone moiety,respectively

Regarding the antiviral mechanism-of-action of PT155 and PT156 versusHCV-1a (isolate H77) HuH-7 cell-derived subgenomic replicon RNAreplication, it is inherently clear that it cannot being mediated byglucocorticoid receptors, since HCV incorporates no DNA stage in itslife cycle. It was recently reported that thiosemicarbazones (DMI-TSC)target bovine viral diarrhea virus type 1 (BVDV-1 strain NADL,Flaviviridae, Pestivirus) NS5B RdRp protein. BVDV-1 is commonly regardedas a suitable surrogate for HCV, because both Flaviviridae polyproteinsequences are closely related (maximal sequence identitiy 32%) (seeAppendix 1). Since the anti-HCV-1a activity of PT155 and PT156 wasdetermined with the HCV-1a (isolate H77) HuH-7 cell-derived subgenomicreplicon, which only codes for the non-structural HCV proteins NS3,NS4A, NS4B, NS5A and NS5B, the inhibiting action of PT155 and PT156 mustbe confined to these five genes and/or gene products (proteins).

Implications for Antiviral Chemotherapy of Hepatitis C Infections:

HCV is one of the major causative agents for chronic hepatitis,cirrhosis and hepatocellular carcinoma. As reported by the WHO, HCVinfects about 2.2% of the world's population, with over a million newcases occurring each year. Furthermore, 27% of these infectedindividuals eventually progress to liver cirrhosis among whom 25%finally develop hepatocellular carcinomas. HCV is a positive-sense RNAvirus that exhibits extensive genetic heterogeneity and a high level ofresistance to antiviral drugs in vivo and in vitro. As such, HCV geneticvariation poses a huge problem for global public health.

As an essence, PT156 successfully targets human hepatitis C virusRNA-dependent RNA polymerase NS5B protein, a “hard” target involvingviral RNA replication [“hard” targets represent indispensable andnon-compensable intracellular aspects of the viral life cycle; “soft”targets represent dispensable and compensable extracellular aspects ofthe viral life cycle, for example attachment/entry/fusion inhibitors (orneuraminidase inhibitors)]. This qualifies PT156 as efficaciousantihepaciviral compound worth further investigation.

TABLE 10 RNA viruses inhibited by PT compounds Virus Reference Virusstrain Cell line drug PT150 PT155 PT156 Caliciviridae, Norovirus 2′-C-Norwalk virus GT1 HG23 methylcytidine (replicon) (RS-446) EC₅₀ (μM) 6.91.1 26.0 >100 EC₉₀ (μM) 18.0 8.1 77.0 >100 CC₅₀ (μM) >300 8.7 111.0 >100SI₅₀ (CC₅₀/EC₅₀) >43 7.9 4.3 — Flaviviridae, Flavivirus 6-azauridineZika virus MR766 HuH-7 EC₅₀ (μM) 13.1 7.4 0.45 6.8 CC₅₀ (μM) >408 7.439.7 51.9 SI₅₀ (CC₅₀/EC₅₀) >31 1.0 88.2 7.6 Picornaviridae, Enterovirus,Enterovirus C pirodavir Poliovirus type 3 WM-3 Vero 76 EC₅₀ (μM) 0.875.6 0.74 51.9 CC₅₀ (μM) >27 7.4 45.4 51.9 SI₅₀ (CC₅₀/EC₅₀) >31 1.3 61.41.0 Picornaviridae, Enterovirus, Enterovirus A pirodavir Enterovirus 71Tainan/ Vero 76 4643/98 EC₅₀ (μM) 0.097 7.4 0.51 51.9 CC₅₀ (μM) >27 7.445.4 51.9 SI₅₀ (CC₅₀/EC₅₀) >278 1.0 89.0 1.0

TABLE 11 Blood Borne viruses (HIV-1, HCV) inhibited by PT CompoundsVirus Virus strain Cell line Reference drug PT150 PT155 PT156Retroviridae, Lentivirus zidovudine (AZT) HIV-1 LAI PBMC (RETROVIR ™)(LAV-1) [averaged from 20 independent determinations (μM ± s.d.)] EC₅₀(μM) 0.0044 ± 0.0039 (n = 20) 8.9 5.5 23.2 EC₉₀ (μM) 0.0299 ± 0.0245 (n= 20) 21.6 16.2 >100 CC₅₀ (μM) PBMC >100 20.6 82.6 73.5 SI₅₀(CC₅₀/EC₅₀) >22,696 (n = 20) 2.3 15.0 3.2 Additional cytotoxicity onmalignant CCRF- cells CEM CC₅₀ (μM) 56.1 15.4 7.1 23.8 Additionalcytotoxicity on kidney Vero cells CC₅₀ (μM) 39.5 44.3 >100 68.6Additional cytotoxicity on primary HFF-1 cells CC₅₀ (μM) — >150 >15013.44 Flaviviridae, Hepacivirus Hepatitis C virus clone B HuH-72′-C-methylcytidine (replicon) (genotype 1a) (RS-446) Inhibition of HCVreplicon RNA synthesis at the 98.08 16.71 59.03 73.91 fixedconcentration of 10 μM of the test compound (%) Inhibition of cellularribosomal RNA (rRNA) 31.09 −39.51 −13.71 3.88 synthesis at the fixedconcentration of 10 μM of the test compound (%) TaqMane ® RT-qPCRthreshold cycle value 5.72 0.26 1.29 1.94 difference (ΔC_(T)) of HCVreplicon RNA synthesis at the fixed concentration of 10 μM of the testcompound TaqMane ® RT-qPCR threshold cycle value 0.54 −0.19 0.06difference (ΔC_(T)) of cellular ribosomal RNA (rRNA) synthesis at thefixed concentration of 10 μM of the test compound

TABLE 12 Compilation of in vitro animal screening Virus Strain Cell LineTreatment Assay Type Conc Unit (+ Range) EC₅₀ CC₅₀ SI₅₀ Norwalk virusNorovirus/1968/US HG23 RS-446 Replicon 3.7-100 μM 6.9 >300 >44 Norwalkvirus Norovirus/1968/US HG23 PT150 Replicon 0.1-100 μM 1.1 8.7 7.9Norwalk virus Norovirus/1968/US HG23 PT155 Replicon 0.1-100 μM 26 1114.3 Norwalk virus Norovirus/1968/US HG23 PT156 Replicon 0.1-100μM >100 >100 1 Chikungunya S27 (VR-67) Vero 76 INFERGEN ™ Neutral red0.00001-0.01 μg/mL 0.00006 >0.01 >167 virus Chikungunya S27 (VR-67) Vero76 INFERGEN ™ Visual 0.00001-0.01 μg/mL 0.00006 >0.01 >167 virusChikungunya S27 (VR-67) Vero 76 PT150 Neutral red 0.1-100 μg/mL >2.6 2.60 virus Chikungunya S27 (VR-67) Vero 76 PT150 Visual 0.1-100 μg/mL >4.24.2 0 virus Chikungunya S27 (VR-67) Vero 76 PT155 Neutral red 0.1-100μg/mL >8.4 8.4 0 virus Chikungunya S27 (VR-67) Vero 76 PT155 Visual0.1-100 μg/mL 15 24 1.6 virus Chikungunya S27 (VR-67) Vero 76 PT156Neutral red 0.1-100 μg/mL >11 11 0 virus Chikungunya S27 (VR-67) Vero 76PT156 Visual 0.1-100 μg/mL 32 32 1 virus Dengue virus New Guinea C HuH-76-Azauridine Neutral red 0.1-100 μg/mL 4.6 21 4.6 type 2 Dengue virusNew Guinea C HuH-7 6-Azauridine Visual 0.1-100 μg/mL 2.8 >100 >36 type 2Dengue virus New Guinea C HuH-7 PT150 Neutral red 0.1-100 μg/mL >2.5 2.50 type 2 Dengue virus New Guinea C HuH-7 PT150 Visual 0.1-100 μg/mL 3.23.2 1 type 2 Dengue virus New Guinea C HuH-7 PT155 Neutral red 0.1-100μg/mL >3.3 3.3 0 type 2 Dengue virus New Guinea C HuH-7 PT155 Visual0.1-100 μg/mL >13 13 0 type 2 Dengue virus New Guinea C HuH-7 PT156Neutral red 0.1-100 μg/mL >7.4 7.4 0 type 2 Dengue virus New Guinea CHuH-7 PT156 Visual 0.1-100 μg/mL 28 28 0 type 2 Enterovirus D68US/KY/14-18953 RD Enviroxime Neutral red 0.1-100 μg/mL 0.1 25 250Enterovirus D68 US/KY/14-18953 RD Enviroxime Visual 0.1-100 μg/mL <0.132 >320 Enterovirus D68 US/KY/14-18953 RD PT150 Neutral red 0.1-100μg/mL >2.6 2.6 0 Enterovirus D68 US/KY/14-18953 RD PT150 Visual 0.1-100μg/mL 3.2 3.2 1 Enterovirus D68 US/KY/14-18953 RD PT155 Neutral red0.1-100 μg/mL >8.1 8.1 0 Enterovirus D68 US/KY/14-18953 RD PT155 Visual0.1-100 μg/mL 3.2 3.2 1 Enterovirus D68 US/KY/14-18953 RD PT156 Neutralred 0.1-100 μg/mL >18 18 0 Enterovirus D68 US/KY/14-18953 RD PT156Visual 0.1-100 μg/mL 32 32 1 Enterovirus 71 Tainan/4643/98 Vero 76Pirodavir Neutral red 0.1-100 μg/mL 0.1 >10 >100 Enterovirus 71Tainan/4643/98 Vero 76 Pirodavir Visual 0.1-100 μg/mL 0.036 >10 >280Enterovirus 71 Tainan/4643/98 Vero 76 PT150 Neutral red 0.1-100μg/mL >2.7 2.7 0 Enterovirus 71 Tainan/4643/98 Vero 76 PT150 Visual0.1-100 μg/mL 3.2 3.2 1 Enterovirus 71 Tainan/4643/98 Vero 76 PT155Neutral red 0.1-100 μg/mL 0.33 92 280 Enterovirus 71 Tainan/4643/98 Vero76 PT155 Visual 0.1-100 μg/mL 0.36 32 89 Enterovirus 71 Tainan/4643/98Vero 76 PT156 Neutral red 0.1-100 μg/mL 27 32 1.2 Enterovirus 71Tainan/4643/98 Vero 76 PT156 Visual 0.1-100 μg/mL 32 32 1 Herpes simplexE-377 Primary HFF-1 Aciclovir CellTiter- 0.048-150 μM 3.85 >150 >39virus type 1 Glo Herpes simplex E-377 Primary HFF-1 PT150 CellTiter-0.048-150 μM >150 >150 1 virus type 1 Glo Herpes simplex E-377 PrimaryHFF-1 PT155 CellTiter- 0.048-150 μM >30 134.15 <4 virus type 1 GloHerpes simplex E-377 Primary HFF-1 PT156 CellTiter- 0.048-150 μM >613.05 <2 virus type 1 Glo Influenza A California/07/20/09 MDCK PT150Neutral red 0.1-100 μg/mL >29 29 0 virus (H1N1) Influenza ACalifornia/07/20/09 MDCK PT150 Visual 0.1-100 μg/mL >37 37 0 virus(H1N1) Influenza A California/07/20/09 MDCK PT155 Neutral red 0.1-100μg/mL 36 > 100 >2.8 virus (H1N1) Influenza A California/07/20/09 MDCKPT155 Visual 0.1-100 μg/mL 32 >100 >3.1 virus (H1N1) Influenza ACalifornia/07/20/09 MDCK PT156 Neutral red 0.1-100 μg/mL 34 62 1.8 virus(H1N1) Influenza A California/07/20/09 MDCK PT156 Visual 0.1-100μg/mL >42 42 0 virus (H1N1) Influenza A California/07/20/09 MDCKRibavirin Neutral red 0.32-320 μg/ml 14 >320 >23 virus (H1N1) InfluenzaA California/07/20/09 MDCK Ribavirin Visual 0.32-320 μg/ml 13 >320 >25virus (H1N1) MERS HCoV-EMC/2012 MA-104 M128533 Neutral red 0.1-100 μg/mL0.8 >100 >125 coronavirus MERS HCoV-EMC/2012 MA-104 M128533 Visual0.1-100 μg/mL 0.86 >100 >116 coronavirus MERS HCoV-EMC/2012 MA-104 PT150Neutral red 0.1-100 μg/mL 3.1 3.9 1.3 coronavirus MERS HCoV-EMC/2012MA-104 PT150 Visual 0.1-100 μg/mL 3.2 3.6 1.1 coronavirus MERSHCoV-EMC/2012 MA-104 PT155 Neutral red 0.1-100 μg/mL >24 24 0coronavirus MERS HCoV-EMC/2012 MA-104 PT155 Visual 0.1-100 μg/mL >18 180 coronavirus MERS HCoV-EMC/2012 MA-104 PT156 Neutral red 0.1-100 μg/mL3.2 9.7 3 coronavirus MERS HCoV-EMC/2012 MA-104 PT156 Visual 0.1-100μg/mL 3.2 8.6 2.7 coronavirus Poliovirus type 3 WM-3 Vero 76 PirodavirNeutral red 0.01-10 μg/mL 0.41 >10 >24 Poliovirus type 3 WM-3 Vero 76Pirodavir Visual 0.01-10 μg/mL 0.32 >10 >31 Poliovirus type 3 WM-3 Vero76 PT150 Neutral red 0.1-100 μg/mL 2.1 3.2 1.5 Poliovirus type 3 WM-3Vero 76 PT150 Visual 0.1-100 μg/mL 2.4 3.2 1.3 Poliovirus type 3 WM-3Vero 76 PT155 Neutral red 0.1-100 μg/mL 0.56 31 55 Poliovirus type 3WM-3 Vero 76 PT155 Visual 0.1-100 μg/mL 0.52 32 62 Poliovirus type 3WM-3 Vero 76 PT156 Neutral red 0.1-100 μg/mL 28 32 1.1 Poliovirus type 3WM-3 Vero 76 PT156 Visual 0.1-100 μg/mL 32 32 1 Respiratory A2 (VR-1540)MA-104 PT150 Neutral red 0.1-100 μg/mL 1.9 4.3 2.3 syncytial virusRespiratory A2 (VR-1540) MA-104 PT150 Visual 0.1-100 μg/mL 2.4 4.2 1.8syncytial virus Respiratory A2 (VR-1540) MA-104 PT155 Neutral red0.1-100 μg/mL 2.8 3.5 1.3 syncytial virus Respiratory A2 (VR-1540)MA-104 PT155 Visual 0.1-100 μg/mL 3.2 3.7 1.2 syncytial virusRespiratory A2 (VR-1540) MA-104 PT156 Neutral red 0.1-100 μg/mL >29 29 0syncytial virus Respiratory A2 (VR-1540) MA-104 PT156 Visual 0.1-100μg/mL 32 32 1 syncytial virus Respiratory A2 (VR-1540) MA-104 RibavirinNeutral red 0.32-320 μg/mL 6.8 >320 >47 syncytial virus Respiratory A2(VR-1540) MA-104 Ribavirin Visual 0.32-320 μg/mL 6.7 >320 >48 syncytialvirus Rift Valley MP-12 Vero 76 PT150 Neutral red 0.1-100 μg/mL >4.1 4.10 fever virus Rift Valley MP-12 Vero 76 PT150 Visual 0.1-100 μg/mL 3.24.2 1.3 fever virus Rift Valley MP-12 Vero 76 PT155 Neutral red 0.1-100μg/mL 6.4 15 2.3 fever virus Rift Valley MP-12 Vero 76 PT155 Visual0.1-100 μg/mL 2.2 8.6 3.9 fever virus Rift Valley MP-12 Vero 76 PT156Neutral red 0.1-100 μg/mL >27 27 0 fever virus Rift Valley MP-12 Vero 76PT156 Visual 0.1-100 μg/mL 10 10 1 fever virus Rift Valley MP-12 Vero 76Ribavirin Neutral red 1-1000 μg/mL 9.7 >1000 >100 fever virus RiftValley MP-12 Vero 76 Ribavirin Visual 1-1000 μg/mL 8.3 >1000 >120 fevervirus Tacaribe virus TRVL11573 Vero PT150 Neutral red 0.1-100 μg/mL 3.13.5 1.1 Tacaribe virus TRVL11573 Vero PT150 Visual 0.1-100 μg/mL 3.2 4.21.3 Tacaribe virus TRVL11573 Vero PT155 Neutral red 0.1-100 μg/mL >3.83.8 0 Tacaribe virus TRVL11573 Vero PT155 Visual 0.1-100 μg/mL >5.9 5.90 Tacaribe virus TRVL11573 Vero PT156 Neutral red 0.1-100 μg/mL >12 12 0Tacaribe virus TRVL11573 Vero PT156 Visual 0.1-100 μg/mL >12 12 0Tacaribe virus TRVL11573 Vero Ribavirin Neutral red 1-1000 μg/mL7.9 >1000 >130 Tacaribe virus TRVL11573 Vero Ribavirin Visual 1-1000μg/mL 7 >1000 >140 Vaccinia virus Copenhagen Primary HFF-1 CidofovirCellTiter- 0.048-150 μM 7.34 >150 >20 Glo Vaccinia virus CopenhagenPrimary HFF-1 PT150 CellTiter- 0.048-150 μM >150 >150 1 Glo Vacciniavirus Copenhagen Primary HFF-1 PT155 CellTiter- 0.048-150 μM >150 >150 1Glo Vaccinia virus Copenhagen Primary HFF-1 PT156 CellTiter- 0.048-150μM >6 13.44 <2 Glo Venez equine TC-83 Vero 76 INFERGEN ™ Neutral red0.00001-0.01 μg/mL <0.00001 >0.01 >1000 enceph virus Venez equine TC-83Vero 76 INFERGEN ™ Visual 0.00001-0.01 μg/mL 0.000012 >0.01 >833 encephvirus Venez equine TC-83 Vero 76 PT150 Neutral red 0.1-100 μg/mL 3.1 3.51.1 enceph virus Venez equine TC-83 Vero 76 PT150 Visual 0.1-100 μg/mL3.2 3.2 1 enceph virus Venez equine TC-83 Vero 76 PT155 Neutral red0.1-100 μg/mL >20 20 0 enceph virus Venez equine TC-83 Vero 76 PT155Visual 0.1-100 μg/mL >28 28 0 enceph virus Venez equine TC-83 Vero 76PT156 Neutral red 0.1-100 μg/mL >16 16 0 enceph virus Venez equine TC-83Vero 76 PT156 Visual 0.1-100 μg/mL 32 32 1 enceph virus Zika virus MR766HuH-7 6-Azauridine Neutral red 0.1-100 μg/mL 11 >100 >9.1 Zika virusMR766 HuH-7 6-Azauridine Visual 0.1-100 μg/mL 3.2 >100 >31 Zika virusMR766 HuH-7 PT150 Neutral red 0.1-100 μg/mL 3 3.3 1.1 Zika virus MR766HuH-7 PT150 Visual 0.1-100 μg/mL 3.2 3.2 1 Zika virus MR766 HuH-7 PT155Neutral red 0.1-100 μg/mL 0.81 7 8.6 Zika virus MR766 HuH-7 PT155 Visual0.1-100 μg/mL 0.32 28 88 Zika virus MR766 HuH-7 PT156 Neutral red0.1-100 μg/mL 21 31 1.5 Zika virus MR766 HuH-7 PT156 Visual 0.1-100μg/mL 4.2 32 7.6 HIV-1 LAI PBMC Zidovudine RT ³H-dTTP 0.00001-100 μM0.00079 >100 >126582 HIV-1 LAI PBMC PT150 RT ³H-dTTP 0.1-100 μM 8.9 20.62.3 HIV-1 LAI PBMC PT155 RT ³H-dTTP 0.1-100 μM 5.5 82.6 15 HIV-1 LAIPBMC PT156 RT ³H-dTTP 0.1-100 μM 23.2 73.5 3.2 Hepatitis B ayw HepAD38Lamivudine ³²P dot blot μM 0.03 — — virus (HBV) Hepatitis B ayw HepAD38PT150 ³²P dot blot Fixed: 10 μM >10 HBV replication virus (HBV) +188.4%Hepatitis B ayw HepAD38 PT155 ³²P dot blot Fixed: 10 μM >10 HBVreplication virus (HBV) +147.4% % inhib % Inhib rRNA replication (tox)Hepatitis C Clone B HuH-7 RS-446 Replicon Fixed: 10 μM 98.08 31.09 virus(HCV) genotype 1a Hepatitis C Clone B HuH-7 PT150 Replicon Fixed: 10 μM16.71 −39.59 virus (HCV) genotype 1a Hepatitis C Clone B HuH-7 PT155Replicon Fixed: 10 μM 59.03 −13.71 virus (HCV) genotype 1a Hepatitis CClone B HuH-7 PT156 Replicon Fixed: 10 μM 73.91 3.88 virus (HCV)genotype 1a — Cytotoxicity PBMC Cycloheximide CellTiter μM — 0.5 controlMTS — Cytotoxicity PBMC PT150 CellTiter μM — 20.6 control MTS —Cytotoxicity PBMC PT155 CellTiter μM — 82.6 control MTS — CytotoxicityPBMC PT156 CellTiter μM — 73.5 control MTS — Cytotoxicity CCRF-CEMCycloheximide CellTiter μM — 0.1 control MTS — Cytotoxicity CCRF-CEMPT150 CellTiter μM — 15.4 control MTS — Cytotoxicity CCRF-CEM PT155CellTiter μM — 7.1 control MTS — Cytotoxicity CCRF-CEM PT156 CellTiterμM — 23.8 control MTS — Cytotoxicity Vero Cycloheximide CellTiter μM —0.2 control MTS — Cytotoxicity Vero PT150 CellTiter μM — 44.3 controlMTS — Cytotoxicity Vero PT155 CellTiter μM — >100 control MTS —Cytotoxicity VeroPT156 CellTiter μM — 68.6 control MTS — CytotoxicityHepG2 Cycloheximide CellTiter μM — 0.6 control MTS Cycloheximide =cellular toxin control — Cytotoxicity Primary HFF-1 PT150 CellTiter- μM— >150 control Glo — Cytotoxicity Primary HFF-1 PT155 CellTiter- μM— >150 control Glo — Cytotoxicity Primary HFF-1 PT156 CollTiter- μM —13.4 control Glo Legend: Conc Unit (+ Range), concentration unit (μM orμg/mL) and concentration range tested; EC₅₀, effective antiviralconcentration 50%; CC₅₀, cytotoxic concentration 50%; SI₅₀ selectivityindex 50% (CC₅₀/EC₅₀); RS-446, 2′-C-methylcytidine; INFERGEN ™,interferon alfacon-1 = consensus interferon-α; CellTiter-Glo,CellTiter-Glo ® Luminescent Cell Viability Assay (Promega) quantifyingATP with ATP-dependent Ultra-Glo ™ Recombinant Luciferase; RT ³H-dTTP,measurement of [5α-³H]dTTP incorporation into poly(rA)•poly(dT) directedby the primed RNA template poly(rA)•oligo(dT) by reverse transcriptase(RT); ³²P dot blot, hybridization assay with EcoRI-digested HBV ayw DNA3.2 kb fragment, phosphorus-32 (³²P)-labeled by DNA polymeraseI-mediated nick translation with [α-³²P]dCTP; cycloheximide, eukaryoticprotein synthesis inhibitor; CellTiter MTS, CellTiter 96 ® AQ_(ueous)One Solution Cell Proliferation Assay (Promega) with tetrazolium saltMTS.

Phosphatidylserine Binding

PS (serine-cephalin) represents an important inner membrane lipid in allhuman cells. PS represents a phospholipid, together withphosphatidylcholine (lecithin) and phosphatidylethanolamine(colamine-cephalin). PS is constructed of1,2-diacylglycerol-3-phospho-L-serine. The1,2-diacylglycerol-3-phosphate is also called phosphatidic acid,therefore the term “phosphatidyl”. PS is normally exposed on human cellsonly in the case of apoptosis (programmed cell death, “voluntary cellsuicide”). Enveloped viruses expose PS on their host-captured lipidbilayer membranes constantly. Enveloped viruses utilize this PS-exposureto evade attacks by the human immune system and to enter phagocyticcells like monocytes/macrophages.

3-O-sn-phosphatidyl-L-serine (PS)

The structure of a typical PS, as shown with stearic acid at position1-O, and docosa-4,7,10,13,16,19-hexaenoic acid at position 2-O. Thisrepresents a major serine-cephalin from bovine brain. Fatty acidcomposition at position 1-O and 2-O is subject of variation, dependinge.g. on cell type. The polar head group (phosphoserine) is negativelycharged. The phosphate anion charge and the cation charge of theammonium group neutralize each other. Therefore, PS is net-negativelycharged at physiological pH 7.4.

Sn-Phosphatidylcholine (PC)

The structure of a typical PC, as shown with stearic acid at position1-O, and linoleic acid at position 2-O. This represents a major lecithinfrom egg yolk and human cell membranes. Fatty acid composition atposition 1-O and 2-O is subject of variation, depending e.g. on celltype. The polar head group (phosphocholine) is zwitterionic. Thephosphate anion charge and the cation charge of the ammonium groupneutralize each other. Therefore, PC is net-neutral at physiological pH7.4. Both ORG34517 and PT155 bind to PS, but not to PC. Weak(stereospecific!) binding to PC is only observed for the PT155-component(E)-ORG34517 thiosemicarbazone, which is contained in the threecomponent mixture PT155. Therefore, the PS-binding of ORG34517 and PT155is selective. ORG34517- and PT155-binding to PS is observed both inPS-micelles and non-micellar PS. The three components of PT155 [the two(E/Z)-ORG34517 thiosemicarbazones, and the residual (original) ORG34517]are readily resolved in the thin-layer chromatographic run.

Most Confirmed PS-Interception-Susceptible Enveloped Viruses are RNAViruses:

Ebola and Marburg virus (Filoviridae)|Ross River virus, chikungunyavirus, Sindbis virus, eastern equine encephalitis virus (Togaviridae,Alphavirus)|vesicular stomatitis virus (Rhabdoviridae,Vesiculovirus)|Amapari virus, Pichindé virus, Tacaribe virus, Juninvirus, Machupo virus (Arenaviridae, Mammarenavirus)|West Nile virus,dengue virus, yellow fever virus (Flaviviridae, Flavivirus)|humanimmunodeficiency virus type 1 (Retroviridae, Lentivirus)|Moloney murineleukemia virus (Retroviridae, Gammaretrovirus)|influenza A virus(Orthomyxoviridae)|respiratory syncytial virus (Paramyxoviridae,Pneumovirinae, Pneumovirus)

Confirmed PS-Interception-Susceptible Enveloped DNA Viruses are:

vaccinia virus (Poxviridae, Chordopoxvirinae, Orthopoxvirus)|herpessimplex virus type 1, herpes simplex virus type 2 (Herpesviridae,Alphaherpesvirinae, Simplexvirus)|human cytomegalovirus (Herpesviridae,Betaherpesvirinae, Cytomegalovirus) Autographa californicanucleopolyhedrovirus (Baculoviridae, Alphabaculoviridae) (an insectvirus)Prospected PS-interception-susceptible important enveloped RNA virusesare (important human viruses are highlighted in red):Ebola and Marburg virus (Filoviridae)|Semliki Forest virus, Ross Rivervirus, chikungunya virus, O'nyong-nyong virus, Sindbis virus,eastern/western/Venezuelan equine encephalitis virus (Togaviridae,Alphavirus)|rubella (German measles) virus (Togaviridae,Rubivirus)|rabies virus, Lagos bat virus, Mokola virus (Rhabdoviridae,Lyssavirus)|Amapari virus, Pichindé virus, Tacaribe virus, Junin virus,Machupo virus, Guanarito virus, Sabia virus, Lassa virus (Arenaviridae,Mammarenavirus)|West Nile virus, dengue virus, yellow fever virus, Zikavirus, Japanese encephalitis virus, St. Louis encephalitis virus,tick-borne encephalitis virus, Omsk hemorrhagic fever virus, KyasanurForest virus (Flaviviridae, Flavivirus)|human hepatitis C virus(Flaviviridae, Hepacivirus)|human immunodeficiency virus type 1(Retroviridae, Lentivirus)|influenza A/B virus (Orthomyxoviridae, thecommon ‘flu’ virus)|respiratory syncytial virus (Paramyxoviridae,Pneumovirinae, Pneumovirus)|Hendra virus, Nipah virus (Paramyxoviridae,Paramyxovirinae, Henipavirus)|measles virus (Paramyxoviridae,Paramyxovirinae, Morbillivirus)Prospected PS-interception-susceptible enveloped DNA viruses are:variola major (smallpox) virus (Poxviridae, Chordopoxvirinae,Orthopoxvirus)|human hepatitis B virus (Hepadnaviridae,Orthohepadnavirus)|hepatitis delta virus (hepatitis D virus) (unassignedFamily, Deltavirus)|herpes simplex virus type 1, herpes simplex virustype 2 (Herpesviridae, Alphaherpesvirinae, Simplexvirus)|humancytomegalovirus (Herpesviridae, Betaherpesvirinae, Cytomegalovirus)

Norovirus

Norovirus infect humans and many other mammals causing severegastroenteritis. Norovirus causes approximately 90% of epidemicnonbacterial outbreaks of gastroenteritis around the world (Lindesmithet al., 2003). Norovirus have also been isolated from numerous otherspecies including pigs, cattle, sheep, and mice. The zoonotic potentialof norovirus is largely unstudied and may represent a reservoir fromwhich new strains may emerge (Thorne & Goodfellow, 2014). Vaccines arenot currently available and treatments which lessen the length ofillness, severity of symptoms, period of infectiousness and loss ofproductivity and life are highly desirable.

The norovirus protein VP1 is unstable during capsid assembly andrequires the host protein Hsp90 to assemble mature viral particles.Vaschist et al. (2012) have demonstrated that geldanamycin binds toHsp90 and inhibits norovirus assembly and budding. Hsp90 complexes withGRα in the host cytosol and is, therefore, susceptible to the action ofhGRα antagonists, such as PT15x compounds according to the predictedstoichiometry:

[hGRα-PT150-(Hsp90)n]m (n=1, 2, . . . ; m=2)

The compounds of the invention have demonstrated activity against bothhuman and murine norovirus, suggesting broader applications among othernorovirus in veterinary medicine, research applications involving miceand human medical applications.

Results: PT150 is highly active versus human Norwalk virus HuH-7-derivedHG23 cell self-replicating RNA replicon replication [Norwalk virus(human norovirus) genotype 1 (GT1), 1968 original isolate, Hu/NV/Norwalkvirus/1968/US (GenBank: M87661.2)].Mechanism-of-action: Heat-shock protein 90 (Hsp90) was suggested astherapeutic target for noroviruses in 2015 by Vashist et al. Themechanism is clearly glucocorticoid receptor-dependent, since PT150, ashigh-affinity human glucocorticoid receptor-alpha (hGRalpha) ligand, ishighly active against norovirus replicon RNA replication, whereas PT155is marginally active and PT156 is inactive. PT150 can reduceavailability of Hsp90 by trapping it in an transcriptionally inactive,dimeric complex:

[hGRalpha-PT150-(Hsp90)n]m (n=1, 2, . . . ; m=2)

The norovirus capsid protein VP1 is a very unstable protein, and must beprotected by the molecular chaperone Hsp90 to survive in the host cellcytosol. If Hsp90 is captured by PT150 in an inactive complex withhGRalpha, then norovirus VP1 is degraded quickly by unknown mechanisms.Vashist et al. used geldanamycin derivatives to prove this. Geldanamycinbinds to Hsp90, also 17-allylamino-17-demethoxygeldanamycin [andwater-soluble derivatives of geldanamycin used in this study like:17-{N-[2-(dimethylamino)ethyl]amino}-17-demethoxygeldanamycin] whichinhibited norovirus assembly and budding.

Taken together, PT150 reduces availability of Hsp90 for noroviruses bytrapping Hsp90 into an inactive complex [hGRalpha-PT150-(Hsp90)n]m (n=1,2, . . . ; m=2). This prevents stabilization of the sensitive viralcapsid protein VP1, and leads to degradation of this viral capsidcomponent by yet unknown mechanisms. In consequence, norovirus assemblyand budding are abrogated by the hGRalpha high-affinity ligand PT150.

Implications for antiviral chemotherapy of Norovirus infections: Theprototype norovirus, Norwalk virus, was first described in 1972 as theetiological agent responsible for an outbreak of acute gastroenteritisin an elementary school in Norwalk, Ohio, USA. Noroviruses are nowaccepted as a leading cause of gastroenteritis in developed anddeveloping countries. Spread primarily via the faecal-oral route,norovirus infections are typically an acute self-limitinggastrointestinal infection. Norovirus gastroenteritis has recently beenidentified as a significant cause of morbidity and mortality in theimmunocompromised, and can result in long-term persistent disease.Norovirus infection has also been associated with a number of moresignificant clinical outcomes: necrotizing enterocolitis, seizures ininfants, encephalopathy, pneumatosis intestinalis and disseminatedintravascular coagulation, to name but a few. In developing countries,an estimated 200,000 deaths in children <5 years of age are thought tobe due to norovirus infections and they have recently been reported asthe second leading cause of gastroenteritis-related deaths in the USA,typically resulting in 797 deaths per annum. Despite their significantimpact, noroviruses remain one of the most poorly characterized groupsof RNA viruses, due largely to the fact that, despite numerous attempts,human noroviruses have yet to be cultured efficiently in cell culture.

Noroviruses are members of the family Caliciviridae of small,positive-sense RNA viruses, which is divided currently into five genera:Vesivirus, Lagovirus, Nebovirus, Sapovirus and Norovirus. Members of thegenera Norovirus and Sapovirus are able to infect humans and causegastroenteritis. The genus Norovirus is subdivided into at least fivegenotypes (GT1-5). Genotypes GT1, GT2 and GT4 infect humans and causeacute gastroenteritis, but noroviruses have also been isolated fromnumerous other species including pigs (GT2), cattle and sheep (GT3), andmice (GT5). More recently, a novel norovirus identified in domestic dogswith diarrhoea has been proposed to represent a new genotype, GT6. Todate, zoonotic norovirus infections have not been reported, but there isclear evidence of the potential for transmission. For example, humannoroviruses can infect gnotobiotic piglets and there is serologicalevidence of human noroviruses in pigs. Whilst antibodies to GT6noroviruses have been identified in veterinarians, it has yet to bedetermined whether infection results in clinical disease. Additionalstudies are required to further elucidate the zoonotic potential ofnoroviruses and whether animals represent a reservoir from which newstrains may emerge.

Addiction

The invention provides Therapeutic Compositions and Methods to MitigateAddiction. The present invention enables PT150 (formerly ORG34517), orits derivatives (e.g. PT-155, PT-156, PT-157) a highly potent andselective corticosteroid antagonist, to prevent the signs and symptomsof physical withdrawal when administered to someone with physicaladdiction at or near the time of withdrawal of the addictive compound.The mechanism underlying this effect is that the central nervous system(CNS) pathways governing physical withdrawal are dependent on cortisolor other glucocorticoid binding. When blockade is established byadministration of PT150 or its derivatives (e.g. PT155, PT156, PT157,PT158, TCY1, combinations thereof, and pharmaceutically acceptable saltsthereof), the pathways fail to function and physical withdrawal symptomsare not produced. The broad field of addiction research has establishedthat corticosteroids appear to play important roles in other aspects ofaddiction behaviors and mechanisms. They have a key role in promoting ashift from recreational (or medicinal in the case of opiates) tocompulsive use, meaning use that is driven by physical addiction. Theterminology of this shift is tricky, but simply put, goal-tracking inanimal models indicates that animals pay more attentional and displaybehavior directly linked to obtaining a rewarding substance. Forexample, in a rat that is pressing a bar to receive food pellets withopiates in them, the rat initially will focus on the food delivery doorand scratch the door, etc. However, when physical addiction occurs withrepeated exposure to the opiates, animals shift their focus and behaviorto the stimuli that predict drug delivery, namely the bar pressing. Thisis called “sign-tracking”.

An analogous human condition would be an alcohol-dependent individual,for example, that initially experiences thoughts and subjective feelingsof reward when drinking alcohol. With continued use of high doses ofalcohol, physical addiction develops. At that point, alcohol cues (whichmay include, for example, drinking buddies, bar signs, emotional stressor tension, the time of day or day of the week when drinking usuallytakes place) provide “reward” to the addict, in a manner similar to theactual drug of abuse. This is a key signal that physical addiction hasoccurred. Abundant animal work and other studies exploring thetranslation of these findings to human subjects, suggest that increasesin circulating corticosteroids, at the least, are highly correlated withsign-tracking.

Dementia

According to the Alzheimer's Association, Dementia is not a specificdisease. It's an overall term that describes a wide range of symptomsassociated with a decline in memory or other thinking skills severeenough to reduce a person's ability to perform everyday activities.Alzheimer's disease accounts for 60 to 80 percent of cases. Vasculardementia, which occurs after a stroke, is the second most commondementia type.

Glucocorticoid receptor activity, stimulated by administration of thesteroid hormone corticosterone, perturbs neuronal function in the rodenthippocampus, a brain region which is critical to learning and theformation of memories in all mammals and which is known to be atrophiedin those with Alzheimer's Disease or Major Depressive Disorder. Workconducted has demonstrated that such effects are dependent on overactivation of glucocorticoid receptors (GRs; result ofhypercortisolemia) and subsequent enhancement of glutamate receptoractivity via effects at the level of transcription and/or trafficking ofreceptor subunits. Indeed, one medication federally approved for thetreatment of Alzheimer's Disease, Namenda® (memantine), is ashort-acting glutamate receptor inhibitor (reviewed in Zhou et al.2016), supporting evidence of a role for these receptors in theinitiation of brain atrophy.

The evidence discussed above also suggests that modulation of GRs mayrepresent a therapeutic target in the treatment of dementing illnesses,as hypothesized by Dhikav and Anand (2007), though clinical research hasnot yet confirmed this possibility and this has not been tested with useof a selective GR antagonist or active agent, such as ORG34517, PT150,PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof. In a mouse model ofAlzheimer's Disease, subchronic treatment with the non-selectiveGR/progesterone receptor antagonist mifepristone prevented losses inepisodic memory. However, the lack pharmacological specificity observedwith mifepristone use does not allow for a clear examination of GRsignaling in this context.

Studies examined effects of selective and competitive inhibition of GRs,with use of PT150 or PT155, on corticosteroid-induced hippocampalneuronal loss using a validated organotypic slice culture model. Slicecultures of rat hippocampus are surgically harvested and maintained invitro for 5 days prior to experimentation. After 5 days, slice culturesare transferred to treatment wells containing corticosterone (0.1-1 μM)or corticosterone vehicle (peanut oil), for a 5 day continuoustreatment. Portions of these cultures, both corticosterone-exposed and-naïve cultures, are also co-exposed to PT150 or PT155 (1-100 nM), incell culture medium for the duration of the 5 days. After 5 days,cultures were either harvested to measure cytotoxicity (propidium iodideuptake and immunoreactivity of neuron-specific nuclear protein) andglutamate receptor density or exposed to the glutamate receptor agonistNMDA for a period of 24-hr. In the latter cultures, measures ofcytotoxicity and receptor density was assessed after NMDA exposure. Itis revealed that 5 day corticosterone exposure, at concentrationsgreater than 1 nM, induced cytoxicity in the cornu ammonis 1 pyramidalcell layer and the co-exposure to PT150 and PT155 attenuate thiscytotoxicity. This cell layer is a region that is critical for theformation of memory in mammals and is known to degenerate in dementias.It is determined that PT150 and PT155 produce effects of cellularintegrity in corticosterone-naïve cultures. Additionally, corticosteroneexposure upregulates NR2B subunits of ionotropic glutamate receptors andmarkedly enhance the neurotoxicity of NMDA itself, demonstrating asteroid hormone receptor-mediated elevation of glutamate signaling in abrain region that is critical to memory formation.

The present Invention provides for method of use claims and compositionof matter formulation claims. In particular embodiments, the inventionprovides a composition of therapeutically effective amount of a GRantagonist (PT150 or PT155) and at least one additional therapeuticagent selected from the group consisting of at least one ofcholinesterase inhibitors (Aricept, Exelon, Razadyne) and memantine(Namenda).

Amyloid beta (Aβ or Abeta) denotes peptides of 36-43 amino acids thatare crucially involved in Alzheimer's disease as the main component ofthe amyloid plaques found in the brains of Alzheimer patients. Thepeptides result from the amyloid precursor protein (APP), which iscleaved by β and γ secretase to yield Aβ. Aβ is formed after sequentialcleavage of the amyloid precursor protein (APP), a transmembraneglycoprotein of undetermined function. APP can be cleaved by theproteolytic enzymes α-, β- and γ-secretase; Aβ protein is generated bysuccessive action of the β and γ secretases. Aβ molecules can aggregateto form flexible soluble oligomers which may exist in several forms.Certain misfolded oligomers (known as “seeds”) can induce other Aβmolecules to also take the misfolded oligomeric form, leading to a chainreaction akin to a prion infection. The seeds or the resulting amyloidplaques are toxic to nerve cells. The other protein implicated inAlzheimer's disease, tau protein, also forms such prion-like misfoldedoligomers, and there is some evidence that misfolded Aβ can induce tauto misfold.

The triboelectric PT155 could form nanomagnets in the brain of MorbusAlzheimer patients, leading to disruption of Aβ amyloid fibrillaraggregates, restoring brain functions to more or lesser extent. SinceAlzheimer fibrills are itself highly structured aggregates withpolarized order inducing an electromagnetic field in the brain. Byinserting a nanomagnetic material into the Aβ supramolecular orderedβ-sheets could disrupt the polarized order (orientation polarization ofpermanent dipoles) of Alzheimer Aβ fibrills making them subject todegradation in the proteasome.

A nanomagnet is a submicrometric system that presents spontaneousmagnetic order (magnetization) at zero applied magnetic field(remanence). The small size of nanomagnets prevents the formation ofmagnetic domains. The magnetization dynamics of sufficiently smallnanomagnets at low temperatures, typically single-molecule magnets,presents quantum phenomena, such as macroscopic spin tunneling. Atlarger temperatures, the magnetization undergoes random thermalfluctuations (superparamagnetism) which present a limit for the use ofnanomagnets for permanent information storage.

Autism

Autism is a neurodevelopmental disorder characterized by impaired socialinteraction, verbal and non-verbal communication, and restricted andrepetitive behavior. Parents usually notice signs in the first two yearsof their child's life. These signs often develop gradually, though somechildren with autism reach their developmental milestones at a normalpace and then regress. The diagnostic criteria require that symptomsbecome apparent in early childhood, typically before age three. Whileautism is highly heritable, researchers suspect both environmental andgenetic factors as causes. In rare cases, autism is strongly associatedwith agents that cause birth defects. Controversies surround otherproposed environmental causes. Autism affects information processing inthe brain by altering how nerve cells and their synapses connect andorganize; how this occurs is not well understood. In the DSM V it is oneof three recognized disorders in the autism spectrum disorders (ASDs),the other two being Asperger syndrome, which lacks delays in cognitivedevelopment and language, and pervasive developmental disorder, nototherwise specified (commonly abbreviated as PDD-NOS), which isdiagnosed when the full set of criteria for autism or Asperger syndromeare not met. The triboelectric PT155 can form nanomagnets in the brainof autism patients, leading to unknown effects of nerve cells (neurons).It is believed that in autism spectrum disorders cortisol levels areelevated

Therapeutic Compositions and Methods for Treating and PreventingTuberculosis

Tuberculosis (TB) is on the top of global infectious disease list,linked to 1.5 million deaths in 2014 and 9.6 million infected. TB isresponsible for 1 in 3 deaths for HIV positive patients, and anestimated 480,000 cases of multidrug resistant TB exist globally (WHO,2016). There are no effective vaccines for TB. Existingchemotherapeutics have extensive side effects, and require prolongedtreatment for success, discounting the possibilities of drug resistance(Gillespie & Murphy, 2011; Korb et al., 2016).

The causative agent, Mycobacterium tuberculosis, utilizes multiplestrategies to manipulate the host immune response to entrench itself andavoid clearance. Following engulfment by macrophage, M. tuberculosisevades destruction by blocking full activation of the phagosome as wellas altering the behaviors of signaling molecules that modulateinflammation and adaptive immunity (Russell, 2007; Sasindran &Torrelles, 2011; Korb et al., 2016). The Toll-like Receptor (TLR) familyis partially responsible for modulating these responses (Hermoso et al.,2004; Coutinho & Chapman, 2011) through cascade-altering reactions usinga counterbalance between the inflammatory effects of tumor necrosisfactor alpha (TNF-α) and the immunosuppressive effects ofglucocorticoids (GC). Antagonism of the GC pathways offers a potentialway to shift the cellular behaviors of infected cells to encourage aprolonged immune response and clearing of TB (Brown et al., 1993;Bottasso, 2007).

Combination Therapy

Compositions and methods for treating GC-responsive conditions,including for example, the prevention or addiction induced anxiety andwithdrawal side effects as a therapeutic, for wound healing andtransplants, for the prevention or treatment of stress inducedosteoporosis and for the rapid healing of bone related injuries, andregenerative therapy, in a subject are provided by the presentinvention.

Methods of treating a GC-responsive condition in a subject are providedaccording to embodiments of the present invention which includesadministering, in combination, a GR antagonist and at least oneadditional therapeutic agent selected from the group consisting of atleast one anti-anxiety drug, at least one anti-depressant drug, and atleast one neuroleptic medication and combinations thereof, wherein theat least one anti-anxiety drug is selected from the group consisting ofalprazolam, bromazepam, diazepam, lorazepam, clonazepam, temazepam,oxazepam, flunitrazepam, triazolam, chlordiazepoxide, flurazepam,estazolam, nitrazepam, and pharmaceutically acceptable salts, isomers,and mixtures thereof; and/or at least one anti-depressant drug selectedfrom the group consisting of citalopram, escitalopram oxalate,fluoxetine, fluvoxamine, paroxetine, sertraline, dapoxetine; venlafaxineand duloxetine; harmaline, iproniazid, isocarboxazid, nialamide,pargyline, phenelzine, selegiline, toloxatone, tranylcypromine,brofaromine, moclobemide; amitriptyline, amoxapine, butriptyline,clomipramine, desipramine, dibenzepin, dothiepin, doxepin, imipramine,iprindole, lofepramine, melitracen, nortriptyline, opipramol,protriptyline, trimipramine; maprotiline, mianserin, nefazodone,trazodone, and pharmaceutically acceptable salts, isomers, andcombinations thereof, and/or at least one neuroleptic drug selected fromthe group consisting of Haloperidol, Droperidol, Benperidol, Triperidol,Melperone, Lenperone, azaperone, Domperidone, risperidone,Chlorpromazine, Fluphenazine, Perphenazine, Prochlorperazine,Thioridazine, Trifluoperazine, Mesoridazine, Periciazine, Promazine,Triflupromazine, Levomepromazine, Promethazine, Pimozide, Cyamemazine,Chlorprothixene, Clopenthixol, Flupenthixol, Thiothixene,Zuclopenthixol, Clozapine, Olanzapine, Risperidone, Quetiapine,Ziprasidone, Amisulpride, Asenapine, Paliperidone, Iloperidone,Zotepine, Sertindole, Lurasidone, Aripiprazole, and pharmaceuticallyacceptable salts, isomers, and combinations thereof, in therapeuticallyeffective amounts. In certain embodiments the agents are administered inthe same dosage form. In certain embodiments the therapeutic agents areadministered separately. In particular embodiments, the inventionprovides a composition a therapeutically effective amount of a GRantagonist and at least one additional therapeutic agent selected fromthe group consisting of at least one antianxiety drug, at least oneanti-depressant drug, and at least one neuroleptic medication andcombinations thereof, wherein the at least one anti-anxiety drug isselected from the group consisting of alprazolam, bromazepam, diazepam,lorazepam, clonazepam, temazepam, oxazepam, flunitrazepam, triazolam,chlordiazepoxide, flurazepam, estazolam, nitrazepam, andpharmaceutically acceptable salts, isomers, and mixtures thereof; and/orat least one anti-depressant drug selected from the group consisting ofcitalopram, escitalopram oxalate, fluoxetine, fluvoxamine, paroxetine,sertraline, dapoxetine; venlafaxine and duloxetine; harmaline,iproniazid, isocarboxazid, nialamide, pargyline, phenelzine, selegiline,toloxatone, tranylcypromine, brofaromine, moclobemide; amitriptyline,amoxapine, butriptyline, clomipramine, desipramine, dibenzepin,dothiepin, doxepin, imipramine, iprindole, lofepramine, melitracen,nortriptyline, opipramol, protriptyline, trimipramine; maprotiline,mianserin, nefazodone, trazodone, and pharmaceutically acceptable salts,isomers, and combinations thereof, and/or at least one neuroleptic drugselected from the group consisting of Haloperidol, Droperidol,Benperidol, Triperidol, Melperone, Lenperone, azaperone, Domperidone,risperidone, Chlorpromazine, Fluphenazine, Perphenazine,Prochlorperazine, Thioridazine, Trifluoperazine, Mesoridazine,Periciazine, Promazine, Triflupromazine, Levomepromazine, Promethazine,Pimozide, Cyamemazine, Chlorprothixene, Clopenthixol, Flupenthixol,Thiothixene, Zuclopenthixol, Clozapine, Olanzapine, Risperidone,Quetiapine, Ziprasidone, Amisulpride, Asenapine, Paliperidone,Iloperidone, Zotepine, Sertindole, Lurasidone, Aripiprazole, andpharmaceutically acceptable salts, isomers, and combinations thereof. Incertain embodiments the agents are in the same dosage form. In certainembodiments the therapeutic agents are in separate dosage forms.

The phrase “administering in combination” as used herein refers to anyform of administration of one or more GR antagonists and at least oneadditional therapeutic agent selected from the group consisting of atleast one anti-anxiety drug, at least one anti-depressant drug, and atleast one neuroleptic medication and combinations thereof, wherein theat least one anti-anxiety drug is selected from the group consisting ofalprazolam, bromazepam, diazepam, lorazepam, clonazepam, temazepam,oxazepam, flunitrazepam, triazolam, chlordiazepoxide, flurazepam,estazolam, nitrazepam, and pharmaceutically acceptable salts, isomers,and mixtures thereof; and/or at least one anti-depressant drug selectedfrom the group consisting of citalopram, escitalopram oxalate,fluoxetine, fluvoxamine, paroxetine, sertraline, dapoxetine; venlafaxineand duloxetine; harmaline, iproniazid, isocarboxazid, nialamide,pargyline, phenelzine, selegiline, toloxatone, tranylcypromine,brofaromine, moclobemide; amitriptyline, amoxapine, butriptyline,clomipramine, desipramine, dibenzepin, dothiepin, doxepin, imipramine,iprindole, lofepramine, melitracen, nortriptyline, opipramol,protriptyline, trimipramine; maprotiline, mianserin, nefazodone,trazodone, and pharmaceutically acceptable salts, isomers, andcombinations thereof, and/or at least one neuroleptic drug selected fromthe group consisting of Haloperidol, Droperidol, Benperidol, Triperidol,Melperone, Lenperone, azaperone, Domperidone, risperidone,Chlorpromazine, Fluphenazine, Perphenazine, Prochlorperazine,Thioridazine, Trifluoperazine, Mesoridazine, Periciazine, Promazine,Triflupromazine, Levomepromazine, Promethazine, Pimozide, Cyamemazine,Chlorprothixene, Clopenthixol, Flupenthixol, Thiothixene,Zuclopenthixol, Clozapine, Olanzapine, Risperidone, Quetiapine,Ziprasidone, Amisulpride, Asenapine, Paliperidone, Iloperidone,Zotepine, Sertindole, Lurasidone, Aripiprazole, and pharmaceuticallyacceptable salts, isomers, and combinations thereof, in therapeuticallyeffective amounts. In certain embodiments the agents are in the samedosage form. In certain embodiments the therapeutic agents are inseparate dosage forms.

Addiction and Withdrawal

The present invention relates to methods of and compositions fortreating and relieving symptoms associated with substance abuse andwithdrawal. The present invention relates to methods of and compositionsfor treating addiction to, for example, alcohol, drugs, caffeine, sugar,food, nicotine, opiates, and/or marijuana, etc.

Substance addiction and abuse is a multi-factorial neurological disease.Over time, repeated exposure to various substances, both endogenous andexogenous, causes modification of the neurotransmission circuits andadaptations in post-receptor signaling cascades. There are severaleffects of this neuronal modification. Among them, there is a reductionin the ability of natural rewards to activate the reward pathwaysleading to depressed motivation and mood and an increased compulsion tocompensate for the physiological change.

While the common perception underlying addiction is that of a “rewardcircuit”, pleasure may not necessarily be a strong enough impetus todrive people towards their addictions. Rather, addictive behavior arisesfrom an intense desire to manage and/or avoid the anxiety that ariseswhen someone is experiencing withdrawal.

Traditional treatments for substance dependency, such as benzodiazepineabuse, have been based upon cognitive-behavioral therapy or drugtherapy, or a combination thereof. Conventional methods of treatmentfail, however, in that they do not address the physiochemical changesthat occur with addiction and dependence. Thus, conventional treatmentsfor controlling withdrawal symptoms and cravings for addictivesubstances have had limited success and often have undesirable sideeffects.

What is therefore needed are improved methods of, and compositions for,preventing addiction to, and physiological dependence upon addictivesubstances. What is also needed is an improved treatment methodology forcontrolling cravings and withdrawal symptoms caused by substance abuse.

Accordingly, the invention provides methods of, and compositions for,preventing addiction to, and physiological dependence upon addictivesubstances. Also provided are methods of and compositions for animproved treatment methodology for controlling cravings and withdrawalsymptoms caused by substance abuse.

The present invention relates to the use of cortisol blockers(glucocorticoid receptor [GR] antagonists) for the prevention oraddiction induced anxiety and withdrawal side effects as a therapeuticand in concert with a diagnostic.

The compounds of the invention may be administered enterally orparenterally. Mixed with pharmaceutically suitable auxiliaries, e.g. asdescribed in the standard reference, Gennaro et al., Remington'sPharmaceutical Sciences. The compounds may be compressed into soliddosage units, such as pills, tablets, or be processed into capsules orsuppositories. By means of pharmaceutically suitable liquids thecompounds can also be applied in the form of a solution, suspension,emulsion, e.g. for use as an injection preparation or eye drops, or as aspray, e.g. for use as a nasal spray. For making dosage units, e.g.tablets, the use of conventional additives such as fillers, colorants,polymeric binders and the like is contemplated. In general anypharmaceutically acceptable additive which does not interfere with thefunction of the active compounds can be used. Suitable carriers withwhich the compositions can be administered include lactose, starch,cellulose derivatives and the like, or mixtures thereof, used insuitable amounts.

The compounds of the invention can be administered orally, topically,intravenously, etc. By means of pharmaceutically suitable liquids thecompounds can be applied in the form of a solution, suspension, oremulsion. The compounds can also be formulated in a patch, ointment orcan be enclosed in a device for local administration to the skin.

The present invention reflects the role of endogenous glucocorticoids(GCs) in withdrawal from substances of abuse and addictive substances(hereafter referred to as “drug” or “drugs”, inclusive of, but notrestricted to, alcohol, nicotine, caffeine, cocaine (including crackcocaine), cannabis, amphetamines (including crystal methamphetamine),opiates and opiate analogues (including heroine, oxycodone, hydrocodone,hydromorphone, methadone), dextromethorphan, benzodiazepines, ecstacy(MDMA), GHB, barbiturates, khat, kratom, PCP, LSD, ketamine, peyote,mescaline, psilocybin, rohypnol, Salvia divinorum, antidepressants,anti-anxiety 5 medications, sleep aids, allergy medications.

Increased circulating levels of GCs may relate to direct elevatingeffects of substances of abuse or from stress-associated GC elevationsin response to neuropsychiatric and physical stresses of withdrawal.

The present invention relates to co-administration of a selective GCreceptor antagonist, such as ORG 34517, PT150, PT155, PT156, PT157,PT158, TCY1, combinations thereof, and pharmaceutically acceptable saltsthereof administered during the active intoxication phase of drug use,prior to drug use, or after cessation of drug use to reduceneuropsychiatric and physical symptoms of withdrawal, such as anxiety,hallucinations, dysphoria, depression, delirium tremens, chills, shakes,tremors, akathisia, restlessness, restless leg syndrome, musculoskeletalaches and pains, cramping, chills, weakness.

The present invention relates to single dose of GC receptor antagonistor sustained administration of GC receptor antagonist for hours, days,weeks, or months for prevention of and/or treatment of symptoms of drugwithdrawal.

The present invention may be considered for co-administration withanti-anxiety drugs and anti-depressant drugs to better control sporadicepisodes, flare-ups of anxiety or depression. Regular co-administrationof the present invention with anti-anxiety and/or anti-depressant drugs.

The present invention may also be used in concert with a diagnostic (forexample, a diagnostic test using saliva, blood, plasma, serum, urine ortears as substrate) for the specific constituent i.e.: alcohol, cocaine,caffeine, nicotine, etc. to monitor the specific level of saidconstituent in the individual to prevent from occurrences of anxiety andwithdrawals.

The present invention may also be used in concert with a diagnostic (forexample, a diagnostic test using saliva, blood, plasma, serum, urine ortears as substrate) for cortisol to determine which individuals haveelevated circulating cortisol or dysregulated cortisol and may thereforebe most likely to benefit from administration of GC receptor antagonist.

The present invention may be packaged for use alone, as a single dose(by prescription or over the counter), as a limited number of timeddoses in packaging designed to specifically guide self-administration,and in combination with drug or cortisol diagnostic test (using saliva,blood, plasma, serum, urine or tears as substrate) forself-administration or administration by health care professional ortechnician.

Androgen Receptor Antagonists

The compositions and methods of the invention may also make use of oneor more androgen receptor antagonists, such as in a combination with theglucocorticoid receptor antagonist of the invention. For example, theinvention provides with at least one glucocorticoid receptor antagonistin combination with at least one androgen receptor antagonist, such asfor example, ARN 509(4-{7-[6-Cyano-5-(trifluoromethyl)-3-pyridinyl]-8-oxo-6-thioxo-5,7-diazaspiro[3.4] oct-5-yl}-2-fluoro-N-methylbenzamide). ARN-509 is a novel androgenreceptor (AR) antagonist for the treatment of castration-resistantprostate cancer (CRPC). ARN-509 inhibits AR nuclear translocation and ARbinding to androgen response elements and, unlike bicalutamide, does notexhibit agonist properties in the context of AR overexpression.

Another exemplary antiadrogen is bicalutamide, which has the chemicalname (R,S)—N-(4-cyano-3-(4-fluorophenylsulfonyl)-2-hydroxy-2-methyl-3-(triflu-oromethyl)propanamide, Flutamide(brand name Eulexin), nilutamide (brand names Anandron and Nilandron)and bicalutamide (brand name Casodex) are nonsteroidal, “pure”antiandrogens; 5-alpha-reductase inhibitors such as finasteride (brandnames Proscar and Propecia), dutasteride (brand name Avodart),bexlosteride, izonsteride, turosteride, and epristeride areantiandrogenic as they prevent the conversion of testosterone todihydrotestosterone (DHT); Spironolactone (brand names Aldactone andSpirotone), a synthetic 17-spirolactone corticosteroid; Cyproteroneacetate (brand names Androcur, Climen, Diane 35, and Ginette 35) is asynthetic steroid, a potent antiandrogen that also possessesprogestational properties. Hydroxyflutamide.

In some embodiments, steroidal or nonsteroidal androgen receptorantagonists include but are not limited to flutamide, hydroxyflutamide,enzalutamide bicalutamide, nilutamide, or hydroxysteroid dehydrogenaseinhibitor.

In one embodiment, the androgen receptor antagonist is enzalutamide(marketed as Xtandi®, Astellas Pharma US, Inc.), also known as andreferred to herein as MDV3100, having the chemical name4-β-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimida-zolidin-1-yl)-2-fluoro-N-methylbenzamide.

The compositions and methods of the invention may also make use of oneor more androgen receptor antagonist, such as in a combination with theglucocorticoid receptor antagonist of the invention. The androgenreceptor antagonist may be selected from the group consisting of, forexample, flutamide, nilutamide, enzalutamide, bicalutamide, ketonazole,abiraterone, abiraterone acetate, orteronel, finasteride, dutasteride,bexlosteride, izonsteride, turosteride, episteride, dexamethasone,prednisone, leuprolide, goserelin, triptorelin, histrelin, estrogen,MDV3100, Cyproterone acetate, Spironolactone, flutamide,hydroxyflutamide, enzalutamide and combinations thereof.

The selective androgen receptor (AR) antagonists embodied herein haveutility for numerous conditions and diseases such as but not limited tomale contraception; treatment of a variety of male hormone-relatedconditions such as hypersexuality and sexual deviation; treatment ofconditions including benign prostatic hyperplasia, acne vugaris,androgenetic alopecia, and hirsutism; purposefully preventing orcounteracting masculinisation in the case of transsexual womenundergoing sex reassignment therapy; an antineoplastic agent andpalliative, adjuvant or neoadjuvant hormonal therapy in prostate cancer;and decreasing the incidence of, halting or causing a regression ofprostate cancer.

Prostate cancer is one of the most common cancers in men around theworld, and is one of the leading causes of cancer death in men in theUnited States. The androgen receptor antagonist drugs, such as flutamideand bicalutamide, were originally designed to avoid the side effects ofHT but androgen agonism was observed for hydroxyfluamide (the activeform of flutamide) and bicalutamide. The compositions of the presentinvention are combinations of GCRAs and, for example, androgen receptorantagonists, which can be used to alleviate any condition associatedwith inappropriate activation of the androgen receptor. In addition toprostate cancer, other examples of such conditions include acne,hirsutism, seborrhoea, excess sebum, and alopecia. In order to exhibitthe therapeutic properties described above, the compounds need to beadministered in a quantity sufficient to inhibit activation of theandrogen receptor. In a typical embodiment, the compounds areadministered topically, which is especially appropriate for hirsutism,alopecia, acne and hyperseborhhea. Androgens, having a profound effecton hair loss, stimulate hair growth by prolonging the growth phase ofthe hair cycle (anagen) and increasing follicle size. Hair growth on thescalp does not require androgens but, paradoxically, androgens arenecessary for balding on the scalp in genetically predisposedindividuals (androgenic alopecia) where there is a progressive declinein the duration of anagen and in hair follicle size. The compositions ofthe invention may also be used topically to decrease seborrheaproduction and more specifically to alleviate hyperseborrhoea (oilyskin), which can be used topically alleviate acne.

Poly(ADP-Ribose) Polymerase (PARP)

The compositions and methods of the invention may also make use of oneor more PARP inhibitors, such as in a combination with theglucocorticoid receptor antagonist of the invention. The poly(ADP-ribose) polymerase (PARP) is also known as poly (ADP-ribose)synthase and poly ADP-ribosyltransferase. PARP catalyzes the formationof mono- and poly (ADP-ribose) polymers which can attach to cellularproteins (as well as to itself) and thereby modify the activities ofthose proteins. The enzyme plays a role in regulation of transcription,cell proliferation, and chromatin remodeling (see D'amours et al.,Biochem., 342: 249268, 1999).

Poly(ADP-ribose)polymerase has an essential role in facilitating DNArepair, controlling RNA transcription, mediating cell death, andregulating immune response. These actions make PARJP inhibitors targetsfor a broad spectrum of disorders. (Virag L., et al., Pharmacol. Rev.2002 54(3):375-429). In various preclinical cancer models and humanclinical trials, PARP inhibitors have been shown to potentiate radiationand chemotherapy by increasing apoptosis of cancer cells, limiting tumorgrowth, decreasing metastasis, and prolonging the survival oftumor-bearing subjects. (WO 2007-084532; Donawho C. K., et al., ClinCancer Res 2007 13(9):2728-37; Kummar S., et al., J Clin Oncol. 200927(16):2705-11).

PARP comprises an N-terminal DNA binding domain, an automodificationdomain, and a C-terminal catalytic domain. Various cellular proteinsinteract with PARP. The N-terminal DNA binding domain contains two zincfinger motifs. Transcription enhancer factor-1 (TEF-1), retinoid Xreceptor a, DNA polymerase a, X-ray repair cross-complementing factor-1(XRCC1) and PARP itself interact with PARP in this domain. Theautomodification domain contains a BRCT motif, one of theprotein-protein interaction modules. This motif is originally found inthe C-terminus of BRCA1 (breast cancer susceptibility protein 1) and ispresent in various proteins related to DNA repair, recombination andcell-cycle checkpoint control. POU— homeodomain-containing octamertranscription factor-1 (Oct-1), YinYang (YY)1, and ubiquitin conjugatingenzyme 9 (ubc9) could interact with this BRCT motif in PARP.

More than 15 members of the PARP family of genes are present in themammalian genome. PARP family proteins and poly(ADP-ribose)glycohydrolase (PARG), which degrades poly(ADP-ribose) to ADP-ribose,are involved in a variety of cell regulatory functions including DNAdamage response and transcriptional regulation and associated withcarcinogenesis and the biology of cancer. Several PARP family proteinshave been identified. Tankyrase has been found as an interacting proteinof telomere regulatory factor 1 (TRF-1) and is involved in telomereregulation. Vault PARP (VPARP) is a component in the vault complex,which acts as a nuclear-cytoplasmic transporter. PARP-2, PARP-3 and2,3,7,8-tetrachlorodibenzo-p-dioxin inducible PARP (TiPARP) have alsobeen identified. Therefore, poly(ADP-ribose) metabolism could be relatedto a variety of cell regulatory functions.

A member of this gene family is PARP-1. The PARP-1 gene product isexpressed at high levels in the nuclei of cells and is dependent uponDNA damage for activation. It is believed that PARP-1 binds to DNAsingle or double-stranded breaks (DSBs) through an amino-terminalDNA-binding domain. The binding activates the carboxy-terminal catalyticdomain and results in the formation of polymers of ADP-ribose on targetmolecules. PARP-1 is itself a target of poly ADP-ribosylation by virtueof a centrally located automodification domain. The ribosylation ofPARP-1 causes dissociation of the PARP-1 molecules from the DNA. Theentire process of binding, ribosylation, and dissociation occurs veryrapidly. It has been suggested that this transient binding of PARP-1 tosites of DNA damage results in the recruitment of DNA repair machineryor may act to suppress the recombination long enough for the recruitmentof repair machinery.

The source of ADP-ribose for the PARP reaction is nicotinamide adenosinedinucleotide (NAD). NAD is synthesized in cells from cellular ATP storesand thus high levels of activation of PARP activity can rapidly lead todepletion of cellular energy stores. It has been demonstrated thatinduction of PARP activity can lead to cell death that is correlatedwith depletion of cellular NAD and ATP pools. PARP activity is inducedin many instances of oxidative stress or during inflammation. Forexample, during reperfusion of ischemic tissues reactive nitric oxide isgenerated and nitric oxide results in the generation of additionalreactive oxygen species including hydrogen peroxide, peroxynitrate, andhydroxyl radical. These latter species can directly damage DNA and theresulting damage induces activation of PARP activity. Frequently, itappears that sufficient activation of PARP activity occurs such that thecellular energy stores are depleted and the cell dies. A similarmechanism is believed to operate during inflammation when endothelialcells and pro-inflammatory cells synthesize nitric oxide, which resultsin oxidative DNA damage in surrounding cells and the subsequentactivation of PARP activity. The cell death that results from PARPactivation is believed to be a major contributing factor in the extentof tissue damage that results from ischemia-reperfusion injury or frominflammation.

PARP (poly-ADP ribose polymerase) participates in a variety ofDNA-related functions including cell proliferation, differentiation,apoptosis, DNA repair and also effects on telomere length and chromosomestability (d'Adda di Fagagna et al., Nature Gen., 23(1): 76-80, 1999).Oxidative stress-induced overactivation of PARP consumes NAD+ andconsequently ATP, culminating in cell dysfunction or necrosis. Thiscellular suicide mechanism has been implicated in the pathomechanism ofcancer, stroke, myocardial ischemia, diabetes, diabetes-associatedcardiovascular dysfunction, shock, traumatic central nervous systeminjury, arthritis, colitis, allergic encephalomyelitis, and variousother forms of inflammation. PARP has also been shown to associate withand regulate the function of several transcription factors.

PARP Inhibitors

Suitable PARP inhibitors for use in the compositions and methods of theinvention include, but are not limited to,4-[[3-[4-(cyclopropanecarbonyl)piperazine-1-carbonyl]-4-fluorophenyl]-methyl]-2H-phthalazin-1-one(Compound B, i.e., Olaparib), 4-iodo-3-nitrobenzamide (Iniparib),2-[(2R)-2-methylpyrrolidin-2-yl]-1H-benzimidazole-4-carboxamide(ABT-888),8-Fluoro-2-{4-[(methylamino)methyl]-phenyl}-1,3,4,5-tetrahydro-6H-azepino-[5,4,3-cd]indol-6-one (AGO 14699), 4-methoxy-carbazole (CEP 9722),2-[4-[(3S)-piperidin-3-yl]phenyl]indazole-7-carboxamide hydrochloride(MK 4827), and 3-aminobenzamide, or a pharmaceutically acceptable saltthereof. In some embodiments, the Glucocorticoid receptor antagonists,e.g., ORG 34517, PT150, PT155, PT156, PT157, PT158, TCY1, combinationsthereof, and pharmaceutically acceptable salts thereof compositiondescribed herein, is administered in combination with a poly ADP-ribosepolymerase (PARP) inhibitor (e.g., BSI201, Olaparib (AZD-2281), ABT-888,AG014699, CEP 9722, MK 4827, KU-0059436 (AZD2281),LT-673,3-aminobenzamide). Other example PARP inhibitors include, i.e.,pharmacological inhibitors of the enzyme poly ADP ribose polymerase(PARP). Suitable PARP inhibitors maybe iniparib, olaparib, rucaparib,veliparib, or CEP 9722.

Current PARP inhibitors in clinical trials include: Iniparib (Sanofi),Olaparib (AstraZeneca), Rucaparib (Pfizer), Veliparib (Abbott), CEP-9722(Cephalon), MK4827 (Merck), BMN-673 (Biomarin), among others.

Treatment Resistant Prostate Cancer

The invention provides a method for treating and/or preventing treatmentresistant prostate cancer in a patient in need of such treatment and/orprevention, comprising: administering to the patient therapeuticallyeffective amounts of a GCR (glucocorticoid receptor) antagonist asexemplified herein, in combination with, for example, a neoplasiatreating agent, including, for example, an androgen receptor (AR)antagonist. Prostate cancer is the most commonly diagnosed cancer in menand the second leading cause of death from cancer in North American andEuropean males. New therapeutic approaches are needed to prevent andtreat advanced and metastatic prostate cancer. Nutritional factors,particularly high intake of protein and calcium, as well as metabolicsyndrome, are known to modify prostate cancer risk and progression, butthe molecular mechanisms linking nutrition to prostate cancer areunknown. There are also links between prostate cancer and bonemetabolism. Osteocalcin (OC), which encodes a vitamin-K dependenthormone predominantly produced by osteoblasts/osteocytes in bone, whichfunctions to regulate energy metabolism, is also ectopically expressedby some prostate cancers that have a propensity to metastasize to bone.Polymorphisms in OC are also associated with prostate cancerprogression. Recent evidence has also identified a correlation betweenthe bone transcription factor Runx2 and advanced stages of prostate andbreast cancer, as evidenced by the effects of depletion of Runx2 by RNAinterference to inhibit migration and invasive properties of the cellsand prevent metastatic bone disease. It is possible that OC secreted bybone may directly target prostate cancer cells. Finally, androgendeprivation therapy is the principal medical therapy for prostatecancer, but androgen ablation often becomes ineffective in controllingprostate cancer progression and castration-resistant metastatic disease,particularly to bone, becomes incurable. There is growing evidence forthe presence of a putative membrane androgen sensing receptor thatmediates the rapid, non-genomic effects of androgens, which also mightbe involved in prostate cancer growth and metastasis. Regardless, cluesto possible new molecular targets to regulate prostate cancer growth andprogression may be discovered from a better understanding of themolecular mechanisms underlying nutritional risk factors, OC effects andandrogen resistance in prostate cancer.

Neoplasia, Cancer, Tumors, Proliferative Diseases, Malignancies andtheir Metastases

The invention provides a method for treating neoplasia in a patient inneed of such treatment, comprising: administering to the patienttherapeutically effective amounts of a GCR (glucocorticoid receptor)antagonist as exemplified herein, such as ORG 34517, PT150, PT155,PT156, PT157, PT158, TCY1, combinations thereof, and pharmaceuticallyacceptable salts thereof, optionally in combination with, for example, aneoplasia treating agent. The term “neoplasia” as used herein refersalso to tumors, proliferative diseases, malignancies and theirmetastases. Examples for cancer diseases are Adenocarcinomas of the headand neck (including salivary glands and oral cavity), gastrointestinaltract (including pharynx, esophagus, stomach, small intestine, largeintestine, anus), lung, liver (including hepatocellular carcinoma,cholangiocarcinoma, and mixed tumors), extrahepatic biliary tract andgallbladder, pancreas (including ductal and acinar types), genitourinarytracts (ovaries, fallopian tubes, endometrium, cervix, and vagina,ureters, urinary bladder, testicles, epididymis, prostate), and skinadnexa; squamous cell carcinomas of the head and neck (includingsalivary glands and oral cavity), gastrointestinal tract (includingpharynx, esophagus, anus), lung, intrahepatic and extrahepatic biliarytree (including gallbladder), pancreas, genitourinary tracts (includingendometrium, cervix, vagina, ureters, urinary bladder, testicles,epididymis, prostate), and skin adnexa; germ cell tumors (includingmalignant teratoma, embryonal carcinoma, struma ovarii, yolk sac tumor,seminoma, choriocarcinoma); sarcomas (including leiomyosarcomas,rhabdomyosarcomas, angiosarcomas, hemangioendotheliomas, liposarcomas,chondosarcomas, fibrosarcomas, Ewing sarcoma, malignant nerve sheathetumors, alveolar soft part sarcomas, clear cell sarcomas, synovialsarcoma, osteosarcomas); malignancies of the central nervous system(including astrocytomas, oligodendroglioma, glioblastoma,medulloblastoma); salivary gland malignancies (including adenoid cysticcarcinoma, adenosquamous carcinoma, clear cell carcinoma,cystadenocarcinoma, mucoepidermoid carcinoma); mixed type carcinomas(including hepatocellular-cholangiocarcinomas, carcinosarcomas, mixedadenoneurondocrine carcinomas, adenosquamous carcinomas); hepatocellularcarcinoma; blastic malignancies (including hepatoblastoma,neuroblastoma, ganglioneuroblastoma, nephroblastoma); renal cellcarcinomas; neuroendocrine carcinomas; thyroid carcinomas (includingpapillary, follicular, medullary, anaplastic carcinomas); parathyroidcarcinomas, pituitary gland carcinomas, adrenal gland carcinomas(including adrenocortical carcinomas, pheochromocytoma), andcombinations thereof.

Cancer Therapies

Any therapy (e.g., therapeutic or prophylactic agent) which is useful,has been used, is currently being used, or may be used for theprevention, treatment and/or management of cancer can be used toprevent, treat, and/or manage the patient whose neoplasia and/or cancerstem cells are monitored in accordance with the methods of theinvention. Also, such neoplasia and/or cancer stem cell monitoring canbe employed in conjunction with any therapy for cancer according to theinstant invention. Therapies (e.g., therapeutic or prophylactic agents)include, but are not limited to, peptides, polypeptides, fusionproteins, nucleic acid molecules, small molecules, mimetic agents,synthetic drugs, inorganic molecules, and organic molecules.Non-limiting examples of cancer therapies include chemotherapies,radiation therapies, hormonal therapies, anti-angiogenesis therapies,targeted therapies, and/or biological therapies includingimmunotherapies and surgery. In certain embodiments, a prophylacticallyand/or therapeutically effective regimen comprises the administration ofa combination of therapies. In certain embodiments, ORG34517, PT150,PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof can be administered as anagent to treat or prevent neoplasia. In certain embodiments, RU486(mifepristone) can be administered as an agent to treat or preventneoplasia.

Examples of cancer therapies include, but are not limited to: acivicin;aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin;altretamine; ambomycin; ametantrone acetate; aminoglutethimide;amsacrine; anastrozole; anthracyclin; anthramycin; asparaginase;asperlin; azacitidine (Vidaza); azetepa; azotomycin; batimastat;benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate;bisphosphonates (e.g., pamidronate (Aredria), sodium clondronate(Bonefos), zoledronic acid (Zometa), alendronate (Fosamax), etidronate,ibandornate, cimadronate, risedromate, and tiludromate); bizelesin;bleomycin sulfate; brequinar sodium; bropirimine; busulfan;cactinomycin; calusterone; caracemide; carbetimer; carboplatin;carmustine; carubicin hydrochloride; carzelesin; cedefingol;chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate;cyclophosphamide; cytarabine (Ara-C); dacarbazine; dactinomycin;daunorubicin hydrochloride; decitabine (Dacogen); demethylation agents,dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel;doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifenecitrate; dromostanolone propionate; duazomycin; edatrexate; eflornithinehydrochloride; EphA2 inhibitors; elsamitrucin; enloplatin; enpromate;epipropidine; epirubicin hydrochloride; erbulozole; esorubicinhydrochloride; estramustine; estramustine phosphate sodium; etanidazole;etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride;fazarabine; fenretinide; floxuridine; fludarabine phosphate;fluorouracil; fluorocitabine; fosquidone; fostriecin sodium;gemcitabine; gemcitabine hydrochloride; histone deacetylase inhibitors(HDAC-Is) hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine;imatinib mesylate (Gleevec, Glivec); interleukin II (includingrecombinant interleukin II, or rIL2), interferon alpha-2a; interferonalpha-2b; interferon alpha-n1; interferon alpha-n3; interferon beta-I a;interferon gamma-I b; iproplatin; irinotecan hydrochloride; lanreotideacetate; lenalidomide (Revlimid); letrozole; leuprolide acetate;liarozole hydrochloride; lometrexol sodium; lomustine; losoxantronehydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride;anti-CD2 antibodies (e.g., siplizumab (MedImmune Inc.; InternationalPublication No. WO 02/098370, which is incorporated herein by referencein its entirety)); megestrol acetate; melengestrol acetate; melphalan;menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine;meturedepa; mifepristone; mitindomide; mitocarcin; mitocromin;mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantronehydrochloride; mycophenolic acid; nocodazole; nogalamycin; ORG 34517;ormaplatin; oxaliplatin; oxisuran; paclitaxel; pegaspargase; peliomycin;pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan;piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium;porfiromycin; prednimustine; procarbazine hydrochloride; puromycin;puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; RU486;safingol; safingol hydrochloride; semustine; simtrazene; sparfosatesodium; sparsomycin; spirogermanium hydrochloride; spiromustine;spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin;tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin;teniposide; teroxirone; testolactone; thiamiprine; thioguanine;thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestoloneacetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate;triptorelin; tubulozole hydrochloride; uracil mustard; uredepa;vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate;vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate;vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicinhydrochloride.

Other examples of cancer therapies include, but are not limited to:20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone;aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TKantagonists; altretamine; ambamustine; amidox; amifostine;aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole;andrographolide; angiogenesis inhibitors; antagonist D; antagonist G;antarelix; anti-dorsalizing morphogenetic protein-I; antiandrogen,prostatic carcinoma; antiestrogen; antineoplaston; antisenseoligonucleotides; aphidicolin glycinate; apoptosis gene modulators;apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; argininedeaminase; asulacrine; atamestane; atrimustine; axinastatin 1;axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatinIII derivatives; balanol; batimastat; BCR/ABL antagonists;benzochlorins; benzoyl staurosporine; beta lactam derivatives;beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor;bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistrateneA; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine;calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2;capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRestM3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinaseinhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins;chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine;clomifene analogues; clotrimazole; collismycin A; collismycin B;combretastatin A4; combretastatin analogue; conagenin; crambescidin 816;crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A;cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate;cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B;deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;diaziquone; didemnin B; didox; diethylnorspermine;dihydro-5-azacytidine; dihydrotaxol, dioxamycin; diphenyl spiromustine;docetaxel; docosanol; dolasetron; doxifluridine; droloxifene;dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine;edrecolomab; eflomithine; elemene; emitefur; epirubicin; epristeride;estramustine analogue; estrogen agonists; estrogen antagonists;etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine;fenretinide; filgrastim; finasteride; flavopiridol; flezelastine;fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex;formestane; fostriecin; fotemustine; gadolinium texaphyrin; galliumnitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine;glutathione inhibitors; HMG CoA reductase inhibitors (e.g.,atorvastatin, cerivastatin, fluvastatin, lescol, lupitor, lovastatin,rosuvastatin, and simvastatin); hepsulfam; heregulin; hexamethylenebisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene;idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod;immunostimulant peptides; insulin-like growth factor-1 receptorinhibitor; interferon agonists; interferons; interleukins; iobenguane;iododoxorubicin; ipomeanol, 4-iroplact; irsogladine; isobengazole;isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F;lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinansulfate; leptol statin; letrozole; leukemia inhibiting factor; leukocytealpha interferon; leuprolide+estrogen+progesterone; leuprorelin; levamisole; LFA-3TIP (Biogen, Cambridge, Mass.; International Publication No.WO 93/0686 and U.S. Pat. No. 6,162,432); liarozole; linear polyamineanalogue; lipophilic disaccharide peptide; lipophilic platinumcompounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol;lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetiumtexaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A;marimastat; masoprocol; maspin; matrilysin inhibitors; matrixmetalloproteinase inhibitors; menogaril; merbarone; meterelin;methioninase; metoclopramide; MIF inhibitor; mifepri stone; miltefosine;mirimostim; mismatched doublestranded RNA; mitoguazone; mitolactol;mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;06-benzylguanine; octreotide; okicenone; oligonucleotides; onapri stone;oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin;oxaunomycin; paclitaxel; paclitaxel analogues; paclitaxel derivatives;palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene;parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfatesodium; pentostatin; pentrozole; perflubron; perfosfamide; perillylalcohol; phenazinomycin; phenylacetate; phosphatase inhibitors;picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetinA; placetin B; plasminogen activator inhibitor; platinum complex;platinum compounds; platinum-triamine complex; porfimer sodium;porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2;proteasome inhibitors; protein A-based immune modulator; protein kinaseC inhibitor; protein kinase C inhibitors, microalgal; protein tyrosinephosphatase inhibitors; purine nucleoside phosphorylase inhibitors;purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene;raf antagonists; raltitrexed; ramosetron; ras farnesyl proteintransferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptinedemethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RIIretinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginoneB1; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim;Sdi 1 mimetics; semustine; senescence derived inhibitor 1; senseoligonucleotides; signal transduction inhibitors; signal transductionmodulators; gamma secretase inhibitors, single chain antigen bindingprotein; sizofuran; sobuzoxane; sodium borocaptate; sodiumphenylacetate; solverol; somatomedin binding protein; sonermin;sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin1; squalamine; stem cell inhibitor; stem-cell division inhibitors;stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactiveintestinal peptide antagonist; suradista; suramin; swainsonine;synthetic glycosaminoglycans; tallimustine; 5-fluorouracil; leucovorin;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;thalidomide; velaresol; veramine; verdins; verteporfin; vinorelbine;vinxaltine; anti-integrin antibodies (e.g., anti-integrin a. sub.vb.sub0.3 antibodies); vorozole; zanoterone; zeniplatin; zilascorb; andzinostatin stimalamer.

A non-limiting list of compounds that could be used to target cancerstem cells includes: inhibitors of interleukin-3 receptor (IL-3R) andCD123 (including peptides, peptide-conjugates, antibodies,antibody-conjugates, antibody fragments, and antibodyfragment-conjugates that target IL-3R or CD123); cantharidin;norcantharidin and analogs and derivatives thereof; Notch pathwayinhibitors including gamma secretase inhibitors; sonichedgehog/smoothened pathway inhibitors including cyclopamine and analogsthereof; antibodies to CD96; certain NF-kB/proteasome inhibitorsincluding parthenolide and analogs thereof; certain triterpenesincluding celastrol; certain mTOR inhibitors; compounds and antibodiesthat target the urokinase receptor; sinefungin; certain inosinemonophosphate dehydrogenase (IMPDH) inhibitors; PPAR-alpha andPPAR-gamma agonists and antagonists (including pioglitazone,tesaslitazar, muraglitazar, peliglitazar, lobeglitazone, balaglitazone,ragaglitazar, rosiglitazone, farglitazar, sodeiglitazar, reglitazar,naveglitazar, oxeglitazar, metaglidasen, netoglitazone, darglitazone,englitazone, thiazolidinediones, aleglitazar, edaglitazone,rivoglitazone, troglitazone, imiglitazar, and sipoglitazar); telomeraseinhibitors; antibodies to EpCAM (ESA); GSK-3 beta agonists andantagonists (including Lithium, 6-bromoinirubin-3′-oxime (BIO), TDZD8);Wnt pathway inhibitors including antibodies to frizzled or smallmolecules that inhibit disheveled/frizzled or beta catenin; anti-CD20antibodies and conjugates (e.g. Rituxan, Bexxar, Zevalin) for novel usein multiple myeloma or melanoma; anti-CD133 antibody; anti-CD44antibody; antibodies to IL-4; certain differentiation agents such asversnarinone; compounds that target CD33 such as an antibody orbetulinic acid; compounds that target lactadherin such as an antibody;small molecules or antibodies that target CXCR4 or SDF-1; smallmolecules or antibodies that target multi-drug resistance pumps;inhibitors of survivin; inhibitors of XIAP; small molecules that targetBcl-2; antibodies to CLL-1; and furin inhibitors (such ascucurbitacins).

An additional non-limiting list of compounds that could also be used totarget cancer and/or cancer stem cells includes: i) antibodies, antibodyfragments, and proteins that are either naked or conjugated to atherapeutic moiety that target certain cell surface targets on cancerstem cells, or ii) small molecules known in the art including ones thatcan be further optimized (e.g., via chemistry) or identified via acancer stem cell-based screen (e.g., such as one that would determinewhether a compound impairs proliferation or viability of a cancer stemcell through standard methods, the cell surface and intracellulartargets including (not meant to be exhaustive) are: Rexl (Zfp42), CTGF,Activin A, Wnt, FGF-2, HIF-1, AP-2gamma, Bmi-1, nucleostemin, hiwi,Moz-TIF2, Nanog, beta-arrestin-2, Oct-4, Sox2, stella, GDF3, RUNX3,EBAF, TDGF-1, nodal, ZFPY, PTNE, Evi-1, Pax3, Mcl-1, c-kit, Lex-1, Zfx,lactadherin, aldehyde dehydrogenase, BCRP, telomerase, CD133, Bcl-2,CD26, Gremlin, and FoxC2.

In some embodiments, the therapy(ies) is an immunomodulatory agent.Non-limiting examples of immunomodulatory agents include proteinaceousagents such as cytokines, peptide mimetics, and antibodies (e.g., human,humanized, chimeric, monoclonal, polyclonal, Fvs, ScFvs, Fab or F(ab)2fragments or epitope binding fragments), nucleic acid molecules (e.g.,antisense nucleic acid molecules and triple helices), small molecules,organic compounds, and inorganic compounds. In particular,immunomodulatory agents include, but are not limited to, methotrexate,leflunomide, cyclophosphamide, cytoxan, Immuran, cyclosporine A,minocycline, azathioprine, antibiotics (e.g., FK506 (tacrolimus)),methylprednisolone (MP), corticosteroids, steroids, mycophenolatemofetil, rapamycin (sirolimus), mizoribine, deoxyspergualin, brequinar,malononitriloamides (e.g., leflunamide), T cell receptor modulators,cytokine receptor modulators, and modulators mast cell modulators. Otherexamples of immunomodulatory agents can be found, e.g., in U.S.Publication No. 2005/0002934 A1 at paragraphs 259-275 which isincorporated herein by reference in its entirety. In one embodiment, theimmunomodulatory agent is a chemotherapeutic agent. In an alternativeembodiment, the immunomodulatory agent is an immunomodulatory agentother than a chemotherapeutic agent. In some embodiments, thetherapy(ies) used in accordance with the invention is not animmunomodulatory agent. In some embodiments, the therapy(ies) is ananti-angiogenic agent. Non-limiting examples of anti-angiogenic agentsinclude proteins, polypeptides, peptides, fusion proteins, antibodies(e.g., human, humanized, chimeric, monoclonal, polyclonal, Fvs, ScFvs,Fab fragments, F(ab).sub.2 fragments, and antigen-binding fragmentsthereof) such as antibodies that specifically bind to TNF-alpha, nucleicacid molecules (e.g., antisense molecules or triple helices), organicmolecules, inorganic molecules, and small molecules that reduce orinhibit angiogenesis. Other examples of anti-angiogenic agents can befound, e.g., in U.S. Publication No. 2005/0002934 A1 at paragraphs277-282, which is incorporated by reference in its entirety. In otherembodiments, the therapy(ies) is not an anti-angiogenic agent.

In certain embodiments, the therapy(ies) is an alkylating agent, anitrosourea, an antimetabolite, and anthracyclin, a topoisomerase IIinhibitor, or a mitotic inhibitor. Alkylating agents include, but arenot limited to, busulfan, cisplatin, carboplatin, chlorambucil,cyclophosphamide, ifosfamide, decarbazine, mechlorethamine, mephalen,and themozolomide. Nitrosoureas include, but are not limited tocarmustine (BCNU) and lomustine (CCNU). Antimetabolites include but arenot limited to 5-fluorouracil, capecitabine, methotrexate, gemcitabine,cytarabine, and fludarabine. Anthracyclins include but are not limitedto daunorubicin, doxorubicin, epirubicin, idarubicin, and mitoxantrone.Topoisomerase II inhibitors include, but are not limited to, topotecan,irinotecan, etopiside (VP-16), and teniposide. Mitotic inhibitorsinclude, but are not limited to taxanes (paclitaxel, docetaxel), and thevinca alkaloids (vinblastine, vincristine, and vinorelbine). In someembodiments of the invention, the therapy(ies) includes theadministration cantharidin or an analog thereof. The invention includesthe use of agents that target cancer stem cells. In certain embodiments,the agent acts alone. In other embodiments, the agent is attacheddirectly or indirectly to another therapeutic moiety. Non-limitingexamples of therapeutic moieties include, but are not limited toalkylating agents, anti-metabolites, plant alkaloids, cytotoxic agents,chemotherapeutic agents (e.g., a steroid, cytosine arabinoside,fluoruracil, methotrexate, aminopterin, mitomycin C, demecolcine,etoposide, mithramycin, calicheamicin, CC-1065, chlorambucil ormelphalan), radionuclides, therapeutic enzymes, cytokines, toxinsincluding plant-derived toxins, fungus-derived toxins, bacteria-derivedtoxin (e.g., deglycosylated ricin A chain, a ribosome inactivatingprotein, alpha-sarcin, aspergillin, restirictocin, a ribonuclease, adiphtheria toxin, Pseudomonas exotoxin, a bacterial endotoxin or thelipid A moiety of a bacterial endotoxin), growth modulators and RNase.In some embodiments, the agent used is an agent that binds to a marker,e.g., an antigen on a cancer stem cell. In a specific embodiment, theagent binds to an antigen that is expressed at a greater level on cancerstem cells than on normal stem cells. In a specific embodiment, theagent binds specifically to a cancer stem cell antigen that is not anormal stem cell. In other embodiments, the therapy(ies) is an agentthat binds to a marker on cancer stem cells. In one embodiment, theagent that binds to a marker on cancer stem cells is an antibody or anantibody conjugated to a therapeutic moiety or an antibody fragmentconjugated to a therapeutic moiety. For example, in a specificembodiment, the agent binds specifically to the IL-3 Receptor (IL-3R).In some embodiments, the agent that binds to the IL-3R is an antibody oran antibody fragment that is specific for IL-3R. In some embodiments,the antibody or antibody fragment is conjugated either chemically or viarecombinant technology to a therapeutic moiety (e.g., a chemotherapeuticagent, a plant-, fungus- or bacteria-derived toxin, a radionuclide)using a linking agent to effect a cell killing response. In certainembodiments, the antibody, antibody-conjugate, antibody fragment, orantibody fragment-conjugate binds to the .alpha.-subunit of IL-3R (i.e.,the CD123 antigen). In other embodiments, the antibody,antibody-conjugate, antibody fragment, or antibody fragment-conjugatebinds to the IL-3R, containing both the .alpha. and .beta. subunits.Methods for preparing antibodies to IL-3R and mimetics of antibodies toIL-3R are described in U.S. Pat. No. 6,733,743 B2, which is incorporatedherein by reference in its entirety.

In other embodiments, the agent that binds to a marker on cancer stemcells is a ligand. In some embodiments, the ligand is a cytokine thatbinds to a cytokine receptor on cancer stem cells. In a particularembodiment, the ligand is interleukin-3 (IL-3) which can be conjugatedto a therapeutic moiety that includes a chemotherapeutic agent, aplant-, fungus-, or bacteria-derived toxin, or a radionuclide. TheIL-3-conjugate prophylactic and/or therapeutic therapy or regimen can bein the form of a recombinant fusion protein in embodiments where theconjugate is a toxin and the toxin is a protein, such as diphtheriatoxin. Methods for preparing and isolating an IL-3-diphtheria toxinfusion protein (IL3DT) are described in Frankel et al., “Diphtheriatoxin fused to human interleukin-3 is toxic to blasts from patients withmyeloid leukemias,” Leukemia 14:576 (2000) and Urieto et al., ProteinExpression and Purification 33: 123-133 (2004), the disclosures of whichare incorporated by reference in their entireties.

In certain embodiments, antibodies or fragments thereof that bind to amarker on cancer stem cells are substantially non-immunogenic in thetreated subject. Methods for obtaining non-immunogenic antibodiesinclude, but are not limited to, chimerizing the antibody, humanizingthe antibody, and isolating antibodies from the same species as thesubject receiving the therapy. Antibodies or fragments thereof that bindto markers in cancer stem cells can be produced using techniques knownin the art. See, for example, paragraphs 539-573 of U.S. Publication No.2005/0002934, which is incorporated by reference in its entirety.

In some embodiments, the therapy comprises the use of X-rays, gamma raysand other sources of radiation to destroy cancer stem cells and/orcancer cells. In specific embodiments, the radiation therapy isadministered as external beam radiation or teletherapy, wherein theradiation is directed from a remote source. In other embodiments, theradiation therapy is administered as internal therapy or brachytherapywherein a radioactive source is placed inside the body close to cancerstem cells, cancer cells and/or a tumor mass.

In some embodiments, the therapy used is a proliferation based therapy.Non-limiting examples of such therapies include a chemotherapy andradiation therapy as described supra. Currently available therapies andtheir dosages, routes of administration and recommended usage are knownin the art and have been described in such literature as the Physician'sDesk Reference (60th ed., 2006).

In a specific embodiment, cycling therapy involves the administration ofa first cancer therapeutic for a period of time, followed by theadministration of a second cancer therapeutic for a period of time,optionally, followed by the administration of a third cancer therapeuticfor a period of time and so forth, and repeating this sequentialadministration, i.e., the cycle in order to reduce the development ofresistance to one of the cancer therapeutics, to avoid or reduce theside effects of one of the cancer therapeutics, and/or to improve theefficacy of the cancer therapeutics.

When two prophylactically and/or therapeutically effective regimens areadministered to a subject concurrently, the term “concurrently” is notlimited to the administration of the cancer therapeutics at exactly thesame time, but rather, it is meant that they are administered to asubject in a sequence and within a time interval such that they can acttogether (e.g., synergistically to provide an increased benefit than ifthey were administered otherwise). For example, the cancer therapeuticsmay be administered at the same time or sequentially in any order atdifferent points in time; however, if not administered at the same time,they should be administered sufficiently close in time so as to providethe desired therapeutic effect, preferably in a synergistic fashion. Thecombination cancer therapeutics can be administered separately, in anyappropriate form and by any suitable route. When the components of thecombination cancer therapeutics are not administered in the samepharmaceutical composition, it is understood that they can beadministered in any order to a subject in need thereof. For example, afirst prophylactically and/or therapeutically effective regimen can beadministered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequentto (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or12 weeks after) the administration of the second cancer therapeutic, toa subject in need thereof. In various embodiments, the cancertherapeutics are administered 1 minute apart, 10 minutes apart, 30minutes apart, less than 1 hour apart, 1 hour apart, 1 hour to 2 hoursapart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hoursto 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart,10 hours to 11 hours apart, 11 hours to 12 hours apart, no more than 24hours apart or no more than 48 hours apart. In one embodiment, thecancer therapeutics are administered within the same office visit. Inanother embodiment, the combination cancer therapeutics are administeredat 1 minute to 24 hours apart.

In a specific embodiment, the combination therapies have the samemechanism of action. In another specific embodiment, the combinationtherapies each have a different mechanism of action.

Treating or Preventing Infection Related to Acute or Chronic Injury orDisease

The invention provides a method for treating or preventing infectionrelated to acute or chronic injury or disease in a patient in need ofsuch treatment, comprising: administering to the patient therapeuticallyeffective amounts of a GCR (glucocorticoid receptor) antagonist asexemplified herein, such as ORG34517, PT150, PT155, PT156, PT157, PT158,TCY1, combinations thereof, and pharmaceutically acceptable saltsthereof, alone or in combination with, for example, another agent. Inparticular, the invention has application to minimize life-threateningcomplications of persons suffering injury to cells, tissues or organsresulting from bums, shock, stroke, heart attack or other physicalevents, including complications from surgical or clinical interventions,as a consequence of trauma. Injured soldiers on the battlefield orcivilians at a natural disaster site or injured from a terrorist attackare situations where such treatment may be useful. The invention appliesto protecting, preserving or stabilizing key organs such as the heartand brain, other neuronal tissues and cells, renal tissue, lung tissue,muscle tissue, liver and other tissues of the body.

In one form, the invention provides a method of reducing injury to thecells, tissues or organs of a body following trauma by administering acomposition to the body following trauma including: (i) a glucocorticoidreceptor antagonist; and optionally (ii) another pharmaceutical agent.

The term “trauma” is used herein in its broadest sense and refers to aserious or critical injury, wound or shock to the body. Trauma may becaused by unexpected physical damage (or injury) to the body as a resultof, for example, transport or industrial accidents, birth, surgery,heart attack, stroke, bums, complications due to surgery or othermedical interventions etc. Trauma may result from injury to a body, bothin a hospital or out of hospital. Trauma is often associated with traumamedicine practiced in hospital (such as in hospital emergency rooms),during emergency transport or at out-of-hospital environments where atrauma has occurred, such as domestic or industrial accidents, transportaccidents, the battlefield, and terrorist attacks. In many cases, traumatherapy may also include resuscitation therapy. Exemplary injuriesinclude, for example, bums, shock, stroke, heart attack or otherphysical events, including complications from surgical or clinicalinterventions, as a consequence of trauma. Injured soldiers on thebattlefield or civilians at a natural disaster site or injured from aterrorist attack are situations where such treatment may be useful. Theinvention provides a method for treating or preventing infection relatedto acute or chronic injury or disease wherein said method comprisesadministering to a patient in need of such therapy at least oneglucocorticoid receptor antagonist in a therapeutically effectiveamount.

The activities of GR agonists and their alteration of cellular functionsare variable, depending on complex intracellular molecular signalingthat are cell and tissue specific. Amongst the cells that haveglucocorticoid receptors are stem and progenitor cells of all tissuesand organs of the body. Thus, binding of such molecules to normative,“in-tissue” stem cells and the progeny of these stem cells, so-called“transit amplifying” progenitor cells, results in variable, cell andtissue specific effects, inhibitory or enhancing of stem and progenitorcell functions, including activation, proliferation, migration anddifferentiation all of which are dependent on the tissue/organ inquestion.

GR antagonists or active agent, such as, for example, ORG 34517, PT150,PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof, will thus block the effectsof GR-agonists in tissue specific fashion, enhancing stem/progenitorcell functioning in some, inhibiting it in others. GR-antagonists willhave beneficial effects in specific clinical settings where regenerativemedicine approaches to disease and wound healing may be of use,including: enhanced post-transplant functioning of autologous stem celltransplants (dependent on tissue of origin and/or target tissue).

Attenuation of the peri-surgical effects of catabolic stress hormonesrelated to surgical or other physical traumas (e.g. combat wounds),wherein the acute or chronic injury or disease is selected from thegroup consisting of vascular events, stroke, cardiac arrest, acute limbinfarction accident/battle field trauma, traumatic limb, hip, braininjuries, post-surgical trauma, major orthopedic, thoracic, abdominal,neurological surgeries.

Systemic GR blockade will be inappropriate, but direct application ofORG 34517, PT150, PT155, PT156, PT157, PT158, TCY1, combinationsthereof, and pharmaceutically acceptable salts thereof to site ofinjury/wounding, either topically (for example, to prevent wounddehiscence) or by direct injection or intravascular infusion (forvisceral organ injuries) will be beneficial.

CNS Injury

The invention provides a method for treating or preventing CNS injury ina patient in need of such treatment, comprising: administering to thepatient therapeutically effective amounts of a GCR (glucocorticoidreceptor) antagonist as exemplified herein, such as ORG34517, PT150,PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof, alone or in combination with,for example, another agent. Conditions suitable for treatment accordingto this invention include, for example, seizure disorders, painsyndromes, neurodegenerative diseases (including motor neuron diseases,myelopathies, radiculopathies, and disorders of the sympathetic nervoussystem), dementias, cerebrovascular conditions, movement disorders,brain trauma, cranial nerve disorders, neuropsychiatric disorders, andother disease neuropathies (including viral associated neuropathies,diabetes associated neuropathies, Guillain-Barre syndrome,dysproteinemias, transthyretin-induced neuropathies, and carpal tunnelsyndrome). As used herein, seizure disorders include complex partialseizures, simple partial seizures, partial seizures with secondarygeneralization, generalized seizures (including absence, grand mal(tonic clonic), status epilepticus, tonic, atonic, myoclonic), neonataland infantile spasms, drug-induced seizures, trauma-induced seizures,and febrile seizures, and additional specific epilepsy syndromes such asjuvenile myoclonic epilepsy, Lennox-Gastaut, mesial temporal lobeepilepsy, nocturnal frontal lobe epilepsy, progressive epilepsy withmental retardation, and progressive myoclonic epilepsy, as well asseizures associated with CNS mass lesions.

Pain syndromes include, for example, headaches (e.g., migraine, tension,and cluster), acute pain, chronic pain, neuropathic pain, nociceptivepain, central pain and inflammatory pain, drug-induced neuropathic pain,causalgia, complex regional pain syndrome types I and II, and reflexsympathetic dystrophy (RSDS).

Neurodegenerative diseases include Alzheimer's disease, Parkinson'sDisease, multiple sclerosis, Huntington's Disease, ALS, spinal muscularatrophy, muscular dystrophies prion-related diseases, cerebellar ataxia,Friedrich's ataxia, SCA, Wilson's disease, RP, Gullian Barre syndrome,Adrenoleukodystrophy, Menke's Sx, cerebral autosomal dominantarteriopathy with subcortical infarcts (CADASIL), Charcot Marie Toothdiseases, neurofibromatosis, von-Hippel Lindau, Fragile X, spasticparaplegia, tuberous sclerosis complex, Wardenburg syndrome, spinalmotor atrophies, Tay-Sachs, Sandoff disease, familial spasticparaplegia, myelopathies, radiculopathies, encephalopathies associatedwith trauma, radiation, drugs and infection, and disorders of thesympathetic nervous system (e.g., Shy Drager (familial dysautonomia),diabetic neuropathy, drug-induced and alcoholic neuropathy).

Dementias include Alzheimer's disease, Parkinson's disease, Pick'sdisease, frontotemporal dementia, vascular dementia, normal pressurehydrocephalus, Huntington's disease, and MCI. Cerebrovascular conditionsamenable to treatment according to the present invention includecerebrovascular disease and strokes (e.g., thrombotic, embolic,thromboembolic, hemorrhagic [including AVM and berry aneurysms],venoconstrictive, and venous).

Included in movement disorders are Parkinson's disease, dystonias,benign essential tremor, tardive dystonia, tardive dyskinesia, andTourette's syndrome. Brain trauma as used herein includes traumaticbrain and spinal cord injuries as well as brain injuries from radiation.Cranial nerve disorders include trigeminal neuropathy, trigeminalneuralgia, Menier's syndrome, glossopharangela neuralgia, dysphagia,dysphonia, cranial nerve palsies and Bell's palsy.

Neuropsychiatric disorders include panic syndrome, general anxietydisorder, phobic syndromes of all types, mania, manic depressiveillness, hypomania, unipolar depression, depression, stress disorders,PTSD, somatoform disorders, personality disorders, psychosis, andschizophrenia), and drug dependence/addiction (e.g., alcohol,psychostimulants (e.g., crack, cocaine, speed, meth), opioids, andnicotine), and drug-induced psychiatric disorders.

Other disease neuropathies that may be treated with the compositions andmethods described herein include Guillain-Barre, diabetes associatedneuropathies, dysproteinemias, transthyretin-induced neuropathies,neuropathy associated with HIV, herpes viruses (including herpes zoster)or other viral infection, neuropathy associated with Lyme disease,carpal tunnel syndrome, tarsal tunnel syndrome, amyloid-inducedneuropathies, leprous neuropathy, Bell's palsy, compressionneuropathies, sarcoidosis-induced neuropathy, polyneuritis cranialis,heavy metal induced neuropathy, transition metal-induced neuropathy,drug-induced neuropathy, postmeningitis syndrome, post-polio syndrome,prion diseases, and radiation associated neuropathic syndromes. Otherdiseases amenable to treatment with the present invention includefatigue syndromes (e.g., chronic fatigue syndrome and fibromyalgia),ataxic syndromes, olivopontoicerebellar degeneration, striatonigraldegeneration, and axonic brain damage.

The present invention is particularly useful in the treatment ofneuropsychiatric disorders such as depression, agitation, anxiety,seizure disorders such as grand mal seizures, status epilepticus,migraine pain treatment and prophylaxis, Alzheimer's disease,Parkinson's disease, and traumatic brain and spinal cord injury.

Also, the higher doses enabled by the present invention are expected tobe of particular importance for dementias including Alzheimer's disease,Parkinson's disease, and vascular dementia, pain syndromes, includingheadaches and migraines, seizure disorders, movement disorders, andbrain trauma.

Furthermore, the ease of use and convenience of a dosage form provideddeveloped to be delivered at once per day or less frequentadministration at a therapeutically effective quantity from the onset oftherapy is of value in the treatment of dementias including Alzheimer'sdisease and Parkinson's disease, seizure disorders, pain syndromes, andcerebrovascular conditions.

Enhanced Memory and/or Performance. Situational stress can lead toelevated circulating levels of cortisol which, in turn, can impair shortterm memory formation. For example, student exam periods in high school,college, as well as graduate school and professional certification andlicensing exams can lead to such stress and, therefore, to aself-defeating, cortisol associated deficit in learning that may impairformation of study-based memories and lead to poorer than expected testresults. The present invention is particularly useful in the treatmentand/or prevention of short term memory performance by, for example,single or sequential dosing with a glucocorticoid blocking compound orcomposition of the invention, before and during the examination studyperiod, that will prevent suppression of short term memory formation andimprove study and subsequent examination performance.

Immunomodulatory Effect

The inventive pharmaceutical composition may additionally contain one ormore auxiliary substances in order to further increase itsimmunomodulatory effect, for example, ORG 34517, PT150, PT155, PT156,PT157, PT158, TCY1, combinations thereof, and pharmaceuticallyacceptable salts thereof alone or in combination with immune systemregulators (e.g., EL-1R antagonist) for prevention of post-traumatic ordisease-associated systemic immunosuppression at high risk for bacterialinfections (e.g., wound infections, pneumonia, colitis, pyelonephritis,hepatic and splenic abscesses) and sepsis. New compositions and methodsare provided which advantageously employ compounds having a newlydefined immune modulating function, or which have the ability to mimicthat immune modulating function, or a combination of such compounds. Forthe purposes of the present disclosure, the terms “immune mimic,”“immune modulating,” “immune modulator,” “immune modulation,” “immunecontrol,” “immune inhibition,” “immune suppressor,” and the like, referin most instances to the newly identified cancer cell growth (i.e.,proliferation) inhibitory effect of the secretory immune system (i.e.,dimeric/polymeric IgA and pentameric IgM) that is mediated by a newlyidentified Poly-Ig receptor or Poly-Ig-like receptor (also classified asan Fc-like receptor), and not to the usual antibody/antigen recognitionbased immune function of the immune system. In this context, the terms“immune modulation” or “immune enhancement” refer especially to themodulation or enhancement of these cell growth inhibitoryimmunoglobulins of the secretory immune system. The term “immune mimic”refers to a substance (e.g., tamoxifen) that can function in a similarmanner to an immunoglobulin inhibitor of cell growth. In some instances,however, reference is also made herein to “natural immune inhibition,”“immune enhancer,” “immune modulator,” “immune system,” “immunetherapy,” and “immune response,” and the like, in which the conventionalmeanings of those terms are intended and the context so indicates,especially when prior art methods, compounds and compositions aredescribed. Hereinafter, an indication has been made in appropriateinstances whether a conventional definition or the “new” meaning, orboth, is intended. A synergistic action of the compounds of theinvention, e.g., ORG 34517, PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof or aderivative thereof as defined according to the present invention, and ofan auxiliary substance, which may be optionally contained in theinventive pharmaceutical composition as described above, is preferablyachieved thereby. Depending on the various types of auxiliarysubstances, various mechanisms can come into consideration in thisrespect. In general, it is possible to use as auxiliary substance anyagent that influences the immune system in the manner of a “dangersignal” (LPS, GP96, etc.) or cytokines, such as GM-CSF, which allow foran immune response produced by the immune-stimulating adjuvant accordingto the invention to be enhanced and/or influenced in a targeted manner.Particularly preferred auxiliary substances are cytokines, such asmonokines, lymphokines, interleukins or chemokines, that further promotethe innate immune response, such as IL-1, IL-1 receptor antagonist,IL-2, EL-3, EL-4, IL-5, IL-6, EL-7, IL-8, EL-9, ELIO, IL-12, EL-13,EL-14, EL-15, EL-16, IL-17, EL-18, IL-19, EL-20, EL-21, EL-22, DL-23,EL-24, IL-25, EL-26, EL-27, EL-28, EL-29, EL-30, EL-31, EL-32, EL-33,INF-alpha, EFN-beta, INF-gamma, GM-CSF, G-CSF, M-CSF, LT-beta orTNF-alpha, growth factors, such as hGH.

The inventive pharmaceutical composition can also additionally oralternatively contain any further compound, which is known to beimmune-stimulating due to its binding affinity (as ligands) to humanToll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,TLR9, TLR10, or due to its binding affinity (as ligands) to murineToll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,TLR9, TLR10, TLR11, TLR12 or TLR13.

Immunoinflammatory Disorder

Another aspect of the present invention is directed to the use of theinventive compound and/or combination as a therapeutic agent for theprophylaxis and/or treatment of immunoinflammatory disorder. The term“immunoinflammatory disorder” encompasses a variety of conditions,including autoimmune diseases, proliferative skin diseases, andinflammatory dermatoses. Immunoinflammatory disorders result in thedestruction of healthy tissue by an inflammatory process, dysregulationof the immune system, and unwanted proliferation of cells. Examples ofimmunoinflammatory disorders are acne vulgaris; acute respiratorydistress syndrome; Addison's disease; allergic rhinitis; allergicintraocular inflammatory diseases, antineutrophil cytoplasmic antibody(ANCA)-associated small-vessel vasculitis; ankylosing spondylitis;arthritis, asthma; atherosclerosis; atopic dermatitis; autoimmunehepatitis; autoimmune hemolytic anemia; autoimmune hepatitis; Behcet'sdisease; Bell's palsy; bullous pemphigoid; cerebral ischemia; chronicobstructive pulmonary disease; cirrhosis; Cogan's syndrome; contactdermatitis; COPD; Crohn's disease; Cushing's syndrome; dermatomyositis;diabetes mellitus; discoid lupus erythematosus; eosinophilic fasciitis;erythema nodosum; exfoliative dermatitis; fibromyalgia; focalglomerulosclerosis; focal segmental glomerulosclerosis; giant cellarteritis; gout; gouty arthritis; graft versus host disease; handeczema; Henoch-Schonlein purpura; herpes gestationis; hirsutism;idiopathic cerato-scleritis; idiopathic pulmonary fibrosis; idiopathicthrombocytopenic purpura; immune thrombocytopenic purpura inflammatorybowel or gastrointestinal disorders, inflammatory dermatoses; lichenplanus; lupus nephritis; lymphomatous tracheobronchitis; macular edema;multiple sclerosis; myasthenia gravis; myositis; nonspecific fibrosinglung disease; osteoarthritis; pancreatitis; pemphigoid gestationis;pemphigus vulgaris; periodontitis; polyarteritis nodosa; polymyalgiarheumatica; pruritus scroti; pruritis/inflammation, psoriasis; psoriaticarthritis; pulmonary histoplasmosis; rheumatoid arthritis; relapsingpolychondritis; rosacea caused by sarcoidosis; rosacea caused byscleroderma; rosacea caused by Sweet's syndrome; rosacea caused bysystemic lupus erythematosus; rosacea caused by urticaria; rosaceacaused by zoster-associated pain; sarcoidosis; scleroderma; segmentalglomerulosclerosis; septic shock syndrome; shoulder tendinitis orbursitis; Sjogren's syndrome; Still's disease; stroke-induced brain celldeath; Sweet's disease; systemic lupus erythematosus; systemicsclerosis; Takayasu's arteritis; temporal arteritis; toxic epidermalnecrolysis; transplant-rejection and transplant-rejection-relatedsyndromes; tuberculosis; type-1 diabetes; ulcerative colitis; uveitis;vasculitis; and Wegener's granulomatosis. As used herein, “non-dermalinflammatory disorders” include, for example, rheumatoid arthritis,inflammatory bowel disease, asthma, and chronic obstructive pulmonarydisease. By “dermal inflammatory disorders” or “inflammatory dermatoses”is meant an inflammatory disorder selected from psoriasis, guttatepsoriasis, inverse psoriasis, pustular psoriasis, erythrodermicpsoriasis, acute febrile neutrophilic dermatosis, eczema, asteatoticeczema, dyshidrotic eczema, vesicular palmoplantar eczema, acnevulgaris, atopic dermatitis, contact dermatitis, allergic contactdermatitis, dermatomyositis, exfoliative dermatitis, hand eczema,pompholyx, rosacea, rosacea caused by sarcoidosis, rosacea caused byscleroderma, rosacea caused by Sweet's syndrome, rosacea caused bysystemic lupus erythematosus, rosacea caused by urticaria, rosaceacaused by zoster-associated pain, Sweet's disease, neutrophilichidradenitis, sterile pustulosis, drug eruptions, seborrheic dermatitis,pityriasis rosea, cutaneous kikuchi disease, pruritic urticarial papulesand plaques of pregnancy, Stevens-Johnson syndrome and toxic epidermalnecrolysis, tattoo reactions, Wells syndrome (eosinophilic cellulitis),reactive arthritis (Reiter's syndrome), bowel-associateddermatosis-arthritis syndrome, rheumatoid neutrophilic dermatosis,neutrophilic eccrine hidradenitis, neutrophilic dermatosis of the dorsalhands, balanitis circumscripta plasmacellularis, balanoposthitis,Behcet's disease, erythema annulare centrifiigum, erythema dyschromicumperstans, erythema multiforme, granuloma annulare, hand dermatitis,lichen nitidus, lichen planus, lichen sclerosus et atrophicus, lichensimplex chronicus, lichen spinulosus, nummular dermatitis, pyodermagangrenosum, sarcoidosis, subcorneal pustular dermatosis, urticaria, andtransient acantholytic dermatosis.

By “proliferative skin disease” is meant a benign or malignant diseasethat is characterized by accelerated cell division in the epidermis ordermis. Examples of proliferative skin diseases are psoriasis, atopicdermatitis, nonspecific dermatitis, primary irritant contact dermatitis,allergic contact dermatitis, basal and squamous cell carcinomas of theskin, lamellar ichthyosis, epidermolytic hyperkeratosis, premalignantkeratosis, acne, and seborrheic dermatitis. As will be appreciated byone skilled in the art, a particular disease, disorder, or condition maybe characterized as being both a proliferative skin disease and aninflammatory dermatosis. An example of such a disease is psoriasis.

Symptoms and signs of inflammation associated with specific conditionsinclude: rheumatoid arthritis:—pain, swelling, warmth and tenderness ofthe involved joints; generalized and morning stiffness;insulin-dependent diabetes mellitus-insulitis; this condition can leadto a variety of complications with an inflammatory component,including:—retinopathy, neuropathy, nephropathy; coronary arterydisease, peripheral vascular disease, and cerebrovascular disease;autoimmune thyroiditis:—weakness, constipation, shortness of breath,puffiness of the face, hands and feet, peripheral edema, bradycardia;multiple sclerosis:—spasticity, blurry vision, vertigo, limb weakness,paresthesias; uveoretinitis:—decreased night vision, loss of peripheralvision; lupus erythematosus:—joint pain, rash, photosensitivity, fever,muscle pain, puffiness of the hands and feet, abnormal urinalysis(hematuria, cylinduria, proteinuria), glomerulonephritis, cognitivedysfunction, vessel thrombosis, pericarditis; scleroderma:—Raynaud'sdisease; swelling of the hands, arms, legs and face; skin thickening;pain, swelling and stiffness of the fingers and knees, gastrointestinaldysfunction, restrictive lung disease; pericarditis; renal failure;other arthritic conditions having an inflammatory component such asrheumatoid spondylitis, osteoarthritis, septic arthritis andpolyarthritis:—fever, pain, swelling, tenderness; other inflammatorybrain disorders, such as meningitis, Alzheimer's disease, AIDS dementiaencephalitis:—photophobia, cognitive dysfunction, memory loss; otherinflammatory eye inflammations, such as retinitis:—decreased visualacuity; inflammatory skin disorders, such as, eczema, other dermatites(e.g., atopic, contact), psoriasis, bums induced by UV radiation (sunrays and similar UV sources):—erythema, pain, scaling, swelling,tenderness; inflammatory bowel disease, such as Crohn's disease,ulcerative colitis:—pain, diarrhea, constipation, rectal bleeding,fever, arthritis; asthma:—shortness of breath, wheezing; other allergydisorders, such as allergic rhinitis:—sneezing, itching, runny noseconditions associated with acute trauma such as cerebral injuryfollowing stroke-sensory loss, motor loss, cognitive loss; heart tissueinjury due to myocardial ischemia:—pain, shortness of breath; lunginjury such as that which occurs in adult respiratory distresssyndrome:—shortness of breath, hyperventilation, decreased oxygenation,pulmonary infiltrates; inflammation accompanying infection, such assepsis, septic shock, toxic shock syndrome:—fever, respiratory failure,tachycardia, hypotension, leukocytosis; other inflammatory conditionsassociated with particular organs or tissues, such as: (i) nephritis(e.g., glomeralonephritis):—oliguria, abnormal urinalysis; (ii) inflamedappendix:—fever, pain, tenderness, leukocytosis; (iii) gout:—pain,tenderness, swelling and erythema of the involved joint, elevated serumand/or urinary uric acid; (iv) inflamed gall bladder:—abdominal pain andtenderness, fever, nausea, leukocytosis; (v) congestive heartfailure:—shortness of breath, rales, peripheral edema; (vi) Type IIdiabetes:—end organ complications including cardiovascular, ocular,renal, and peripheral vascular disease; (vii) lung (pulmonary)fibrosis:—hyperventilation, shortness of breath, decreased oxygenation;(viii) vascular disease, such as atherosclerosis and restenosis:—pain,loss of sensation, diminished pulses, loss of function; and (ix)alloimmunity leading to transplant rejection:—pain, tenderness, fever.

Neurodegenerative Disease

Another aspect of the present invention is directed to the use of theinventive compound and/or combination as a therapeutic agent for theprophylaxis and/or treatment of Neurodegenerative Disease. Exemplaryactive agents include, for example, ORG34517, PT150, PT155, PT156,PT157, PT158, TCY1, combinations thereof, and pharmaceuticallyacceptable salts thereof.

The present invention also relates generally to the fields of neurologyand psychiatry and to methods of protecting the cells of a mammaliancentral nervous system from damage or injury. Injuries or trauma ofvarious kinds to the central nervous system (CNS) or the peripheralnervous system (PNS) can produce profound and long-lasting neurologicaland/or psychiatric symptoms and disorders. One form that this can takeis the progressive death of neurons or other cells of the centralnervous system (CNS), i.e., neurodegeneration or neuronal degeneration.Neuronal degeneration as a result of, for example; Alzheimer's disease,multiple sclerosis, cerebral-vascular accidents (CVAs)/stroke, traumaticbrain injury, spinal cord injuries, degeneration of the optic nerve,e.g., ischemic optic neuropathy or retinal degeneration and othercentral nervous system disorders is an enormous medical and publichealth problem by virtue of both its high incidence and the frequency oflong-term sequelae. Animal studies and clinical trials have shown thatamino acid transmitters (especially glutamate), oxidative stress andinflammatory reactions contribute strongly to cell death in theseconditions. Upon injury or upon ischemic insult, damaged neurons releasemassive amounts of the neurotransmitter glutamate, which is excitotoxicto the surrounding neurons. Glutamate is a negatively charged amino acidthat is an excitatory synaptic transmitter in the mammalian nervoussystem. Although the concentration of glutamate can reach the millimolarrange in nerve terminals its extracellular concentration is maintainedat a low level to prevent neurotoxicity. It has been noted thatglutamate can be toxic to neurons if presented at a high concentration.The term “excitotoxicity” has been used to describe the cytotoxic effectthat glutamate (and other such excitatory amino acids) can have onneurons when applied at high dosages.

Patients with injury or damage of any kind to the central (CNS) orperipheral (PNS) nervous system including the retina may benefit fromneuroprotective methods. This nervous system injury may take the form ofan abrupt insult or an acute injury to the nervous system as in, forexample, acute neurodegenerative disorders including, but not limitedto; acute injury, hypoxia-ischemia or the combination thereof resultingin neuronal cell death or compromise. Acute injury includes, but is notlimited to, traumatic brain injury (TBI) including, closed, blunt orpenetrating brain trauma, focal brain trauma, diffuse brain damage,spinal cord injury, intracranial or intravertebral lesions (including,but not limited to, contusion, penetration, shear, compression orlaceration lesions of the spinal cord or whiplash shaken infantsyndrome).

In addition, deprivation of oxygen or blood supply in general can causeacute injury as in hypoxia and/or ischemia including, but not limitedto, cerebrovascular insufficiency, cerebral ischemia or cerebralinfarction (including cerebral ischemia or infarctions originating fromembolic occlusion and thrombosis, retinal ischemia (diabetic orotherwise), glaucoma, retinal degeneration, multiple sclerosis, toxicand ischemic optic neuropathy, reperfusion following acute ischemia,perinatal hypoxic-ischemic injury, cardiac arrest or intracranialhemorrhage of any type (including, but not limited to, epidural,subdural, subarachnoid or intracerebral hemorrhage).

Trauma or injury to tissues of the nervous system may also take the formof more chronic and progressive neurodegenerative disorders, such asthose associated with progressive neuronal cell death or compromise overa period of time including, but not limited to, Alzheimer's disease,Pick's disease, diffuse Lewy body disease, progressive supranuclearpalsy (Steel-Richardson syndrome), multisystem degeneration (Shy-Dragersyndrome), chronic epileptic conditions associated withneurodegeneration, motor neuron diseases (amyotrophic lateralsclerosis), multiple sclerosis, degenerative ataxias, cortical basaldegeneration, ALS-Parkinson's-dementia complex of Guam, subacutesclerosing panencephalitis, Huntington's disease, Parkinson's disease,synucleinopathies (including multiple system atrophy), primaryprogressive aphasia, striatonigral degeneration, Machado-Joseph diseaseor spinocerebellar ataxia type 3 and olivopontocerebellar degenerations,bulbar and pseudobulbar palsy, spinal and spinobulbar muscular atrophy(Kennedy's disease), primary lateral sclerosis, familial spasticparaplegia, Werdnig-Hoffmann disease, Kugelberg-Welander disease,Tay-Sach's disease, Sandhoff disease, familial spastic disease,Wohlfart-Kugelberg-Welander disease, spastic paraparesis, progressivemultifocal leukoencephalopathy, familial dysautonomia (Riley-Daysyndrome) or prion diseases (including, but not limited toCreutzfeld-Jakob disease, Gerstmann-Strussler-Scheinker disease, Kurudisease or fatal familial insomnia).

In addition, trauma and progressive injury to the nervous system cantake place in various psychiatric disorders, including but not limitedto, progressive, deteriorating forms of bipolar disorder orschizoaffective disorder or schizophrenia, impulse control disorders,obsessive compulsive disorder (OCD), behavioral changes in temporal lobeepilepsy and personality disorders. In one preferred embodiment thecompounds and/or compositions of the invention would be used to provideneuroprotection in disorders involving trauma and progressive injury tothe nervous system in various psychiatric disorders. These disorderswould be selected from the group consisting of; schizoaffectivedisorder, schizophrenia, impulse control disorders, obsessive compulsivedisorder (OCD) and personality disorders.

In addition, trauma and injury make take the form of disordersassociated with overt and extensive memory loss including, but notlimited to, neurodegenerative disorders associated with age-relateddementia, vascular dementia, diffuse white matter disease (Binswanger'sdisease), dementia of endocrine or metabolic origin, dementia of headtrauma and diffuse brain damage, dementia pugilistica or frontal lobedementia, including but not limited to Pick's Disease.

Other disorders associated with neuronal injury include, but are notlimited to, disorders associated with chemical, toxic, infectious andradiation injury of the nervous system including the retina, injuryduring fetal development, prematurity at time of birth, anoxic-ischemia,injury from hepatic, glycemic, uremic, electrolyte and endocrine origin,injury of psychiatric origin (including, but not limited to,psychopathology, depression or anxiety), injury from peripheral diseasesand plexopathies (including plexus palsies) or injury from neuropathy(including neuropathy selected from multifocal, sensory, motor,sensory-motor, autonomic, sensory-autonomic or demyelinatingneuropathies (including, but not limited to Guillain-Barre syndrome orchronic inflammatory demyelinating polyradiculoneuropathy) or thoseneuropathies originating from infections, inflammation, immunedisorders, drug abuse, pharmacological treatments, toxins, trauma(including, but not limited to compression, crush, laceration orsegmentation traumas), metabolic disorders (including, but not limitedto, endocrine or paraneoplastic), Charcot-Marie-Tooth disease(including, but not limited to, type 1a, 1b, 2,4a or 1-X linked),Friedreich's ataxia, metachromatic leukodystrophy, Refsum's disease,adrenomyeloneuropathy, ataxia-telangiectasia, Djerine-Sottas (including,but not limited to, types A or B), Lambert-Eaton syndrome or disordersof the cranial nerves).

Further indications are cognitive disorders. The term “cognitivedisorder” shall refer to anxiety disorders, delirium, dementia, amnesticdisorders, dissociative disorders, eating disorders, mood disorders,schizophrenia, psychotic disorders, sexual and gender identitydisorders, sleep disorders, somatoform disorders, acute stress disorder,obsessive-compulsive disorder, panic disorder, posttraumatic stressdisorder, specific phobia, social phobia, substance withdrawal delirium,Alzheimer's disease, Creutzfeldt-Jakob disease, head trauma,Huntington's disease, HTV disease, Parkinson's disease, Pick's disease,learning disorders, motor skills disorders, developmental coordinationdisorder, communication disorders, phonological disorder, pervasivedevelopmental disorders, Asperger's disorder, autistic disorder,childhood disintegrative disorder, Rett's disorder, pervasivedevelopmental disorder, attention-deficit/hyperactivity disorder (ADHD),conduct disorder, oppositional defiant disorder, pica, ruminationdisorder, tic disorders, chronic motor or vocal tic disorder, Tourette'sdisorder, elimination disorders, encopresis, enuresis, selective mutism,separation anxiety disorder, dissociative amnesia, depersonalizationdisorder, dissociative fugue, dissociative identity disorder, anorexianervosa, bulimia nervosa, bipolar disorders, schizophreniform disorder,schizoaffective disorder, delusional disorder, psychotic disorder,shared psychotic disorder, delusions, hallucinations, substance-inducedpsychotic disorder, orgasmic disorders, sexual pain disorders,dyspareunia, vaginismus, sexual dysfunction, paraphilias, dyssomnias,breathing-related sleep disorder, circadian rhythm sleep disorder,hypersomnia, insomnia, narcolepsy, dyssomnia, parasomnias, nightmaredisorder, sleep terror disorder, sleepwalking disorder, parasomnia, bodydysmorphic disorder, conversion disorder, hypochondriasis, paindisorder, somatization disorder, alcohol related disorders, amphetaminerelated disorders, caffeine related disorders, cannabis relateddisorders, cocaine related disorders, hallucinogen related disorders,inhalant related disorders, nicotine related disorders, opioid relateddisorders, phencyclidine-related disorder, abuse, persisting amnesticdisorder, intoxication, withdrawal.

The term “bipolar and clinical disorders” shall refer to adjustmentdisorders, anxiety disorders, delirium, dementia, amnestic and othercognitive disorders, disorders usually first diagnosed in infancy(e.g.), childhood, or adolescence, dissociative disorders (e.g.dissociative amnesia, depersonalization disorder, dissociative fugue anddissociative identity disorder), eating disorders, factitious disorders,impulse-control disorders, mental disorders due to a general medicalcondition, mood disorders, other conditions that may be a focus ofclinical attention, personality disorders, schizophrenia and otherpsychotic disorders, sexual and gender identity disorders, sleepdisorders, somatoform disorders, substance-related disorders,generalized anxiety disorder (e.g. acute stress disorder, posttraumaticstress disorder), panic disorder, phobia, agoraphobia,obsessive-compulsive disorder, stress, acute stress disorder, anxietyneurosis, nervousness, phobia, posttraumatic stress disorder,posttraumatic stress disorder (PTSD), abuse, obsessive-compulsivedisorder (OCD), manic depressive psychosis, specific phobias, socialphobia, adjustment disorder with anxious features.

Examples for disorders usually first diagnosed in infancy, childhood, oradolescence are: mental retardation, learning disorders, mathematicsdisorder, reading disorder, disorder of written expression, motor skillsdisorders, developmental coordination disorder, communication disorders,expressive language disorder, phonological disorder, mixedreceptive-expressive language disorder, stuttering, pervasivedevelopmental disorders, Asperger's disorder, autistic disorder,childhood disintegrative disorder, Rett's disorder, pervasivedevelopmental disorder, attention-deficit/hyperactivity disorder (ADHD),conduct disorder, oppositional defiant disorder, feeding disorder ofinfancy or early childhood, pica, rumination disorder, tic disorders,chronic motor or vocal tic disorder, Tourette's syndrome, eliminationdisorders, encopresis, enuresis, selective mutism, separation anxietydisorder, reactive attachment disorder of infancy or early childhood,stereotypic movement disorder.

Examples for substance-related disorders are: alcohol related disorders,amphetamine related disorders, caffeine related disorders, cannabisrelated disorders, cocaine related disorders, hallucinogen relateddisorders, inhalant related disorders, nicotine related disorders,opioid related disorders, psychotic disorder, psychotic disorder,phencyclidine-related disorder, abuse, persisting amnestic disorder,anxiety disorder, persisting dementia, dependence, intoxication,intoxication delirium, mood disorder, psychotic disorder, withdrawal,withdrawal delirium, sexual dysfunction, sleep disorder.

The term “neuroprotection” as used herein shall mean; inhibiting,preventing, ameliorating or reducing the severity of the dysfunction,degeneration or death of nerve cells, axons or their supporting cells inthe central or peripheral nervous system of a mammal, including a human.This includes the treatment or prophylaxis of a neurodegenerativedisease; protection against excitotoxicity or ameliorating the cytotoxiceffect of a compound (for example, a excitatory amino acid such asglutamate; a toxin; or a prophylactic or therapeutic compound thatexerts an immediate or delayed cytotoxic side effect including but notlimited to the immediate or delayed induction of apoptosis) in a patientin need thereof.

The term “a patient in need of treatment with a neuroprotective drug” asused herein will refer to any patient who currently has or may developany of the above syndromes or disorders, or any disorder in which thepatient's present clinical condition or prognosis could benefit fromproviding neuroprotection to prevent the development, extension,worsening or increased resistance to treatment of any neurological orpsychiatric disorder.

The term “treating” or “treatment” as used herein, refers to any indiciaof success in the prevention or amelioration of an injury, pathology orcondition, including any objective or subjective parameter such asabatement; remission; diminishing of symptoms or making the injury,pathology, or condition more tolerable to the patient; slowing in therate of degeneration or decline; making the final point of degenerationless debilitating; or improving a subject's physical or mentalwell-being. The treatment or amelioration of symptoms can be based onobjective or subjective parameters; including the results of a physicalexamination, neurological examination, and/or psychiatric evaluations.

In some embodiments this invention provides methods of neuroprotection.In certain embodiments, these methods comprise administering atherapeutically effective amount of the composition and/or combinationof the invention to a patient who has not yet developed overt, clinicalsigns or symptoms of injury or damage to the cells of the nervous systembut who may be in a high risk group for the development of neuronaldamage because of injury or trauma to the nervous system or because ofsome known predisposition either biochemical or genetic or the findingof a verified biomarker of one or more of these disorders.

Thus, in some embodiments, the methods and compositions of the presentinvention are directed toward neuroprotection in a subject who is atrisk of developing neuronal damage but who has not yet developedclinical evidence. This patient may simply be at “greater risk” asdetermined by the recognition of any factor in a subject's, or theirfamilies, medical history, physical exam or testing that is indicativeof a greater than average risk for developing neuronal damage.Therefore, this determination that a patient may be at a “greater risk”by any available means can be used to determine whether the patientshould be treated with the methods of the present invention.Accordingly, in an exemplary embodiment, subjects who may benefit fromtreatment by the methods and the composition and/or combination of thisinvention can be identified using accepted screening methods todetermine risk factors for neuronal damage. These screening methodsinclude, for example, conventional work-ups to determine risk factorsincluding but not limited to: for example, head trauma, either closed orpenetrating, CNS infections, bacterial or viral, cerebrovascular diseaseincluding but not limited to stroke, brain tumors, brain edema,cysticercosis, porphyria, metabolic encephalopathy, drug withdrawalincluding but not limited to sedative-hypnotic or alcohol withdrawal,abnormal perinatal history including anoxia at birth or birth injury ofany kind, cerebral palsy, learning disabilities, hyperactivity, historyof febrile convulsions as a child, history of status epilepticus, familyhistory of epilepsy or any seizure related disorder, inflammatorydisease of the brain including lupis, drug intoxication either direct orby placental transfer, including but not limited to cocaine poisoning,parental consanguinity, and treatment with medications that are toxic tothe nervous system including psychotropic medications.

The determination of which patients may benefit from treatment with aneuroprotective drug in patients who have no clinical signs or symptomsmay be based on a variety of “surrogate markers” or “biomarkers”.

As used herein, the terms “surrogate marker” and “biomarker” are usedinterchangeably and refer to any anatomical, biochemical, structural,electrical, genetic or chemical indicator or marker that can be reliablycorrelated with the present existence or future development of neuronaldamage. In some instances, brain-imaging techniques, such as computertomography (CT), magnetic resonance imaging (MM) or positron emissiontomography (PET), can be used to determine whether a subject is at riskfor neuronal damage. Suitable biomarkers for the methods of thisinvention include, but are not limited to: the determination by MRI, CTor other imaging techniques, of sclerosis, atrophy or volume loss in thehippocampus or overt mesial temporal sclerosis (MTS) or similar relevantanatomical pathology; the detection in the patient's blood, serum ortissues of a molecular species such as a protein or other biochemicalbiomarker, e.g., elevated levels of ciliary neurotrophic factor (CNTF)or elevated serum levels of a neuronal degradation product; or otherevidence from surrogate markers or biomarkers that the patient is inneed of treatment with a neuroprotective drug.

It is expected that many more such biomarkers utilizing a wide varietyof detection techniques will be developed in the future. It is intendedthat any such marker or indicator of the existence or possible futuredevelopment of neuronal damage, as the latter term is used herein, maybe used in the methods of this invention for determining the need fortreatment with the compounds and methods of this invention.

A determination that a subject has, or may be at risk for developing,neuronal damage would also include, for example, a medical evaluationthat includes a thorough history, a physical examination, and a seriesof relevant bloods tests. It can also include an electroencephalogram(EEG), CT, MRI or PET scan. A determination of an increased risk ofdeveloping neuronal damage or injury may also be made by means ofgenetic testing, including gene expression profiling or proteomictechniques. For psychiatric disorders that may be stabilized or improvedby a neuroprotective drug, e.g., bipolar disorder, schizoaffectivedisorder, schizophrenia, impulse control disorders, etc. the above testsmay also include a present state exam and a detailed history of thecourse of the patients symptoms such as mood disorder symptoms andpsychotic symptoms over time and in relation to other treatments thepatient may have received over time, e.g., a life chart. These and otherspecialized and routine methods allow the clinician to select patientsin need of therapy using the methods and formulations of this invention.In some embodiments of the present invention compounds and/orcompositions suitable for use in the practice of this invention will beadministered either singly or concomitantly with at least one or moreother compounds or therapeutic agents, e.g., with other neuroprotectivedrugs or antiepileptic drugs, anticonvulsant drugs. In theseembodiments, the present invention provides methods to treat or preventneuronal injury in a patient. The method includes the step of;administering to a patient in need of treatment, an effective amount ofthe compounds and/or compositions disclosed herein in combination withan effective amount of one or more other compounds or therapeutic agentsthat have the ability to provide neuroprotection or to treat or preventseizures or epileptogenesis or the ability to augment theneuroprotective effects of the compounds of the invention.

As used herein the term “combination administration” of a compound,therapeutic agent or known drug with the combination of the presentinvention means administration of the drug and the one or more compoundsat such time that both the known drug and/or combination will have atherapeutic effect. In some cases this therapeutic effect will besynergistic. Such concomitant administration can involve concurrent(i.e. at the same time), prior, or subsequent administration of the drugwith respect to the administration of the composition and/or combinationof the present invention. A person of ordinary skill in the art wouldhave no difficulty determining the appropriate timing, sequence anddosages of administration for particular drugs of the present invention.The said one or more other compounds or therapeutic agents may beselected from compounds that have one or more of the followingproperties: antioxidant activity; NMDA receptor antagonist activity,augmentation of endogenous GABA inhibition; NO synthase inhibitoractivity; iron binding ability, e.g., an iron chelator; calcium bindingability, e.g., a Ca (II) chelator; zinc binding ability, e.g., a Zn (II)chelator; the ability to effectively block sodium or calcium ionchannels, or to open potassium or chloride ion channels in the CNS of apatient.

Heart and Vascular Disease

Another aspect of the present invention is directed to the use of theinventive compound and/or combination such as for example, ORG34517,PT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof, as a therapeutic agent forthe prophylaxis and/or treatment of heart disease. Heart disease is ageneral term used to describe many different heart conditions. Forexample, coronary artery disease, which is the most common heartdisease, is characterized by constriction or narrowing of the arteriessupplying the heart with oxygen-rich blood, and can lead to myocardialinfarction, which is the death of a portion of the heart muscle. Heartfailure is a condition resulting from the inability of the heart to pumpan adequate amount of blood through the body. Heart failure is not asudden, abrupt stop of heart activity but, rather, typically developsslowly over many years, as the heart gradually loses its ability to pumpblood efficiently. Risk factors for heart failure include coronaryartery disease, hypertension, valvular heart disease, cardiomyopathy,disease of the heart muscle, obesity, diabetes, and/or a family historyof heart failure.

Examples of cardiovascular diseases and disorders are: aneurysm, stableangina, unstable angina, angina pectoris, angioneurotic edema, aorticvalve stenosis, aortic aneurysm, arrhythmia, arrhythmogenic rightventricular dysplasia, arteriosclerosis, arteriovenous malformations,atrial fibrillation, Behcet syndrome, bradycardia, cardiac tamponade,cardiomegaly, congestive card iomyopathy, hypertrophic cardiomyopathy,restrictive cardiomyopathy, carotid stenosis, cerebral hemorrhage,Churg-Strauss syndrome, diabetes, Ebstein's Anomaly, Eisenmengercomplex, cholesterol embolism, bacterial endocarditis, fibromusculardysplasia, congenital heart defects, heart diseases, congestive heartfailure, heart valve diseases, heart attack, epidural hematoma,hematoma, subdural, Hippel-Lindau disease, hyperemia, hypertension,pulmonary hypertension, cardiac hypertrophy, left ventricularhypertrophy, right ventricular hypertrophy, hypoplastic left heartsyndrome, hypotension, intermittent claudication, ischemic heartdisease, Klippel-Trenaunay-Weber syndrome, lateral medullary syndrome,long QT syndrome mitral valve prolapse, moyamoya disease, mucocutaneouslymph node syndrome, myocardial infarction, myocardial ischemia,myocarditis, pericarditis, peripheral vascular diseases, phlebitis,polyarteritis nodosa, pulmonary atresia, Raynaud disease, Sneddonsyndrome, superior vena cava syndrome, syndrome X, tachycardia,Takayasu's arteritis, hereditary hemorrhagic telangiectasia,telangiectasis, temporal arteritis, tetralogy of Fallot, thromboangiitisobliterans, thrombosis, thromboembolism, tricuspid atresia, varicoseveins, vascular diseases, vasculitis, vasospasm, ventricularfibrillation, Williams syndrome, peripheral vascular disease, varicoseveins and leg ulcers, deep vein thrombosis, Wolff-Parkinson-Whitesyndrome.

Vascular diseases are often the result of decreased perfusion in thevascular system or physical or biochemical injury to the blood vessel.

Peripheral vascular disease (PVD) is defined as a disease of bloodvessels often encountered as narrowing of the vessels of the limbs.There are two main types of these disorders, functional disease whichdoesn't involve defects in the blood vessels but rather arises fromstimuli such as cold, stress, or smoking, and organic disease whicharises from structural defects in the vasculature such asatherosclerotic lesions, local inflammation, or traumatic injury. Thiscan lead to occlusion of the vessel, aberrant blood flow, and ultimatelyto tissue ischemia.

One of the more clinically significant forms of PVD is peripheral arterydisease (PAD). PAD is often treated by angioplasty and implantation of astent or by artery bypass surgery. Clinical presentation depends on thelocation of the occluded vessel. For example, narrowing of the arterythat supplies blood to the intestine can result in severe postprandialpain in the lower abdomen resulting from the inability of the occludedvessel to meet the increased oxygen demand arising from digestive andabsorptive processes. In severe forms the ischemia can lead tointestinal necrosis. Similarly, PAD in the leg can lead to intermittentpain, usually in the calf, that comes and goes with activity. Thisdisorder is known as intermittent claudication (IC) and can progress topersistent pain while resting, ischemic ulceration, and even amputation.

Peripheral vascular disease is also manifested in atheroscleroticstenosis of the renal artery, which can lead to renal ischemia andkidney dysfunction.

One disease in which vascular diseases and their complications are verycommon is diabetes mellitus. Diabetes mellitus causes a variety ofphysiological and anatomical irregularities, the most prominent of whichis the inability of the body to utilize glucose normally, which resultsin hyperglycemia. Chronic diabetes can lead to complications of thevascular system which include atherosclerosis, abnormalities involvinglarge and medium size blood vessels (macroangiopathy) and abnormalitiesinvolving small blood vessels (microangiopathy) such as arterioles andcapillaries. Patients with diabetes mellitus are at increased risk ofdeveloping one or more foot ulcers as a result of established long-termcomplications of the disease, which include impaired nerve function(neuropathy) and/or ischemia. Local tissue ischemia is a keycontributing factor to diabetic foot ulceration.

In addition to large vessel disease, patients with diabetes sufferfurther threat to their skin perfusion in at least two additional ways.First, by involvement of the non-conduit arteries, which aredetrimentally affected by the process of atherosclerosis, and secondly,and perhaps more importantly, by impairment of the microcirculatorycontrol mechanisms (small vessel disease). Normally, when a body partsuffers some form of trauma, the body part will, as part of the body'shealing mechanism, experience an increased blood flow. When small vesseldisease and ischemia are both present, as in the case of many diabetics,this natural increased blood flow response is significantly reduced.This fact, together with the tendency of diabetics to form blood clots(thrombosis) in the microcirculatory system during low levels of bloodflow, is believed to be an important factor in ulcer pathogenesis.Neuropathy is a general term which describes a disease process whichleads to the dysfunction of the nervous system, and is one of the majorcomplications of diabetes mellitus, with no well-established therapiesfor either its symptomatic treatment or for prevention of progressivedecline in nerve function.

The thickening and leakage of capillaries caused by diabetes primarilyaffect the eyes (retinopathy) and kidneys (nephropathy). The thickeningand leakage of capillaries caused by diabetes are also associated withskin disorders and disorders of the nervous system (neuropathy).

The eye diseases associated with diabetes are nonproliferative diabeticretinopathy, proliferative diabetic retinopathy, diabetic maculopathy,glaucoma, cataracts and the like. Other diseases, although not known tobe related to diabetes are similar in their physiological effects on theperipheral vascular system. Such diseases include Raynaud syndrome,CREST syndrome, autoimmune diseases such as erythematosis, rheumatoiddisease, and the like.

As used herein, the term “peripheral vascular diseases” comprises anyperipheral vascular disease including peripheral and autonomicneuropathies. Examples of “peripheral vascular disease” includeperipheral arterial disease, such as chronic arterial occlusionincluding arteriosclerosis, arteriosclerosis obliterans andthromboangiiti s obliterans (Buerger's disease), macroangiopathy,microangiopathy, diabetes mellitus, thrombophlebitis, phlebemphraxis,Raynaud's disease, Raynaud's syndrome, CREST syndrome, health hazard dueto vibration, Sudeck's syndrome, intermittent claudication, cold sensein extremities, abnormal sensation in extremities, sensitivity to thecold, Meniere's disease, Meniere's syndrome, numbness, lack ofsensation, anesthesia, resting pain, causalgia (burning pain),disturbance of peripheral circulation function, disturbance of nervefunction, disturbance of motor function, motor paralysis, diabeticperipheral circulation disorder, lumbar spinal canal stenosis, diabeticneuropathy, shock, autoimmune disease such as erythematosis, rheumatoiddisease and rheumatoid arthritis, autonomic neuropathy, diabeticautonomic neuropathy, autonomic imbalance, orthostatic hypotension,erectile dysfunction, female sexual dysfunction, retrograde ejaculation,cystopathy, neurogenic bladder, defective vaginal lubrication, exerciseintolerance, cardiac denervation, heat intolerance, gustatory sweating,diabetic complication, hyperglycemia, hypoglycemia unawareness,hypoglycemia unresponsiveness; glaucoma, neovascular glaucoma, cataract,retinopathy, diabetic retinopathy, diabetic maculopathy, occlusion ofretinal artery, obstruction of central artery of retina, occlusion ofretinal vein, macular edema, aged macular degeneration, aged disciformmacular degeneration, cystoid macular edema, palpebral edema, retinaledema, chorioretinopathy, neovascular maculopathy, uveitis, iritis,retinal vasculitis, endophthalmitis, panophthalmitis, metastaticophthalmia, choroiditis, retinal pigment epithelitis, conjunctivitis,cyclitis, scleritis, episcleritis, optic neuritis, retrobulbar opticneuritis, keratitis, blepharitis, exudative retinal detachment, cornealulcer, conjunctival ulcer, chronic nummular keratitis, Thygesonkeratitis, progressive Mooren's ulcer, damage of skin, skin ulcerincluding foot ulcer, diabetic ulcer, bum ulcer, lower leg ulcer,postoperative ulcer, traumatic ulcer, ulcer after herpes zoster,radiation ulcer, drug induced ulcer, frostbite (cold injury), chilblain,gangrene and sudden gangrene, angina pectoris/variant angiitis, coronaryarteriosclerosis (chronic ischemic heart disease, asymptomatic ischemicheart disease, arteriosclerotic cardiovascular disease), myocardialinfarction, heart failure, congestive heart failure and painlessischemic heart disease, pulmonary edema, hypertension, pulmonaryhypertension; portal hypertension, diabetic nephropathy, decubitus,renal failure.

Formulations

The compounds and compositions of the invention, such as for example,ORG34517, PT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof,and pharmaceutically acceptable salts thereof, may be administered at adose of less than 400 mg/day. In some embodiments, the compounds andcompositions of the invention are administered at a dose of about 1mg/day, about 2 mg/day, about 5 mg/day, about 10 mg/day, about 15mg/day, about 20 mg/day, about 25 mg/day, about 30 mg/day, about 35mg/day, about 40 mg/day, about 45 mg/day, about 50 mg/day, about 60mg/day, about 70 mg/day, about 80 mg/day, about 90 mg/day, about 100mg/day, about 120 mg/day, about 125 mg/day, about 140 mg/day, about 150mg/day, about 160 mg/day, about 175 mg/day, about 180 mg/day, about 190mg/day, about 200 mg/day, about 225 mg/day, about 250 mg/day, about 275mg/day, about 300 mg/day, about 325 mg/day, about 350 mg/day, about 375mg/day, about 400 mg/day, about 425 mg/day, about 450 mg/day, about 475mg/day, or about 500 mg/day. In certain embodiments, the compounds ofthe invention are administered at a dose of less than 1 mg/day, lessthan 2 mg/day, less than 5 mg/day, less than 10 mg/day, less than 15mg/day, less than 20 mg/day, less than 25 mg/day, less than 30 mg/day,less than 35 mg/day, less than 40 mg/day, less than 45 mg/day, less than50 mg/day, less than 60 mg/day, less than 70 mg/day, less than 80mg/day, less than 90 mg/day, less than 100 mg/day, less than 120 mg/day,less than 125 mg/day, less than 140 mg/day, less than 150 mg/day, lessthan 160 mg/day, less than 175 mg/day, less than 180 mg/day, less than190 mg/day, less than 200 mg/day, less than 225 mg/day, less than 250mg/day, less than 275 mg/day, less than 300 mg/day, less than 325mg/day, less than 350 mg/day, less than 375 mg/day, less than 400mg/day, less than 425 mg/day, less than 450 mg/day, less than 475mg/day, or less than 500 mg/day. In some embodiments, the compounds ofthe invention are administered at a dose of more than 1 mg/day, morethan 2 mg/day, more than 5 mg/day, more than 10 mg/day, more than 15mg/day, more than 20 mg/day, more than 25 mg/day, more than 30 mg/day,more than 35 mg/day, more than 40 mg/day, more than 45 mg/day, more than50 mg/day, more than 60 mg/day, more than 70 mg/day, more than 80mg/day, more than 90 mg/day, more than 100 mg/day, more than 120 mg/day,more than 125 mg/day, more than 140 mg/day, more than 150 mg/day, morethan 160 mg/day, more than 175 mg/day, more than 180 mg/day, more than190 mg/day, more than 200 mg/day, more than 225 mg/day, more than 250mg/day, more than 275 mg/day, more than 300 mg/day, more than 325mg/day, more than 350 mg/day, more than 375 mg/day, more than 400mg/day, more than 425 mg/day, more than 450 mg/day, more than 475mg/day, or more than 500 mg/day.

The compounds and compositions of the invention may be administeredenterally or parenterally. Mixed with pharmaceutically suitableauxiliaries, e.g., as described in the standard reference, Gennaro etal., Remington's Pharmaceutical Sciences. The compounds may becompressed into solid dosage units, such as pills, tablets, or beprocessed into capsules or suppositories. By means of pharmaceuticallysuitable liquids the compounds can also be applied in the form of asolution, suspension, emulsion, e.g. for use as an injection preparationor eye drops, or as a spray, e.g. for use as a nasal spray.

For making dosage units, e.g., tablets, the use of conventionaladditives such as fillers, colorants, polymeric binders and the like iscontemplated, hi general, any pharmaceutically acceptable additive whichdoes not interfere with the function of the active compounds can beused. Suitable carriers with which the compositions can be administeredinclude lactose, starch, cellulose derivatives and the like, or mixturesthereof, used in suitable amounts.

The activities of GR agonists and their alteration of cellular functionsare variable, depending on complex intracellular molecular signalingthat are cell and tissue specific. Amongst the cells that haveglucocorticoid receptors are stem and progenitor cells of all tissuesand organs of the body. Thus, binding of such molecules to normative,“in-tissue” stem cells and the progeny of these stem cells, so-called“transit amplifying” progenitor cells, results in variable, cell andtissue specific effects, inhibitory or enhancing of stem and progenitorcell functions, including activation, proliferation, migration anddifferentiation all of which are dependent on the tissue/organ inquestion.

GR antagonists or active agents, such as, for example, ORG 34517, PT150,PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof, will thus block the effectsof GR-agonists in tissue specific fashion, enhancing stem/progenitorcell functioning in some, inhibiting it in others. GR-antagonists willhave beneficial effects in specific clinical settings where regenerativemedicine approaches to disease and wound healing may be of use,including: enhanced post-transplant functioning of autologous stem celltransplants (dependent on tissue of origin and/or target tissue).

Attenuation of the peri-surgical effects of catabolic stress hormonesrelated to surgical or other physical traumas (e.g. combat wounds),wherein the acute or chronic injury or disease is selected from thegroup consisting of vascular events, stroke, cardiac arrest, acute limbinfarction accident/battle field trauma, traumatic limb, hip, braininjuries, post-surgical trauma, major orthopedic, thoracic, abdominal,neurological surgeries.

Systemic GR blockade will be inappropriate, but direct application ofORG 34517, PT150, PT155, PT156, PT157, PT158, TCY1, combinationsthereof, and pharmaceutically acceptable salts thereof to site ofinjury/wounding, either topically (for example, to prevent wounddehiscence) or by direct injection or intravascular infusion (forvisceral organ injuries) will be beneficial.

Cortisol

The estimated daily cortisol production rate in normal subjects variesbetween 4-15 mg/m.sup.2 per day or, according to more recent studiesbetween 9 and 11 mg/m.sup.2 per day. In order to describe the 24-hourvariation in serum cortisol levels adequately, the day may be dividedinto, for example, four phases. Phase 1 is a 6-hours period of minimalsecretory activity 4 h before and 2 h after onset of sleep. Phase 2refers to the 3rd to 5th hours of sleep when there is a preliminarynocturnal secretory episode. Phase 3 is a 4-hour main secretory phaseduring the last 3 h of sleep and the first hour after wakening. Phase 4is an 11-hour phase of intermittent secretory activity when there is aslow decline in serum levels of cortisol.

In a study by Mah et al. (Clinical Endocrinology (2004) 61, 367-375) thecircadian rhythm of serum cortisol of normal subjects is described. Peaklevels of about 400-800 mmol/1, about 150-300 mmol/1 and about 150mmol/1 are observed at about 6 am, 2 pm and 9 pm, respectively, and thelowest level is about midnight. In this study it is observed that theendogenous cortisol levels reach their highest levels within 30 minutesafter wake-up. In order to mimic the circadian rhythm, Mah et al.recommend a thrice-daily treatment regimen of hydrocortisone, the firstdose taken in the fasted state and delaying the breakfast 1-3 hours andthe other two doses taken 15-60 min before food. A trice-daily regimenis also recommended in a recent review by Czock et al. (Clin.Pharmacokinet (2005) 44, 61-98) due to the short half-life ofhydrocortisone, and for prednisolone a twice-daily regimen is preferredover a once-daily regimen.

Cortisol Test

The absence of rapid response and inexpensive testing for cortisol has,heretofore, prevented the linking of GCR antagonists or active agents(e.g., ORG 34517, PT150, PT155, PT156, PT157, PT158, TCY1, combinationsthereof, and pharmaceutically acceptable salts thereof) to a cortisolpre-test for entry into clinical trials for GCR antagonists and willinhibit the ability to select the patients most likely to receive thebenefit of treatment with the compounds when available for clinical use.The invention provides the pairing of an affordable, real-time cortisoltest (e.g., PopTest Cortisol) which will enable the successfulcompletion of clinical trials for this class of drugs as well as formthe basis for their future, anticipated therapeutic use(s).

Conditions that may be treated using, for example, a linked salivarycortisol quantification test and GCR antagonist or active agent (e.g.,ORG 34517, PT150, PT155, PT156, PT157, PT158, TCY1, combinationsthereof, and pharmaceutically acceptable salts thereof) system include,but are not limited to the following:

Major Depressive Disorder (MDD).

MDD is a psychiatric disorder which has a lifetime prevalence of around8%. One of the most consistent findings in psychiatry is that patientswith major depression present with alterations in thehypothalamic-pituitary-adrenal (HPA) axis. A significant percentage ofdepressed patients exhibit hypersecretion cortisol, as manifested byelevated plasma, cerebrospinal fluid, and salivary concentrations ofcortisol and increased urinary free cortisol. In addition, manydepressed patients exhibit a clear inability to switch off endogenouscortisol release following exogenous challenge with the potent syntheticglucocorticoid dexamethasone (the so-called dexamethasonenon-suppressors) (Gold P. W., et al., Clinical and biochemicalmanifestations of depression: relation to neurobiology of stress. NewEngland J. Med. 319, 413-420, 1988). This ‘sub-group’ of severelycompromised patients are most often the ones in whom depression becomesa life-threatening illness that warrants hospitalization.

Other abnormalities of the HPA axis found in depressed patients areincreased cortisol response to corticotrophin, a blunted corticotrophinresponse to CRH (corticotrophin releasing hormone), and adrenal andpituitary enlargement (for a review see Holsboer, F. and Barden, N.:Endocrine Reviews 1996, 17, 187-205).

These observations have been interpreted to suggest a causalrelationship between disturbed functioning of the HPA axis and thepathology of depression (Murphy, B. E. P. J. of Steroid Biochem. andMol. Biol. 1991, 38, 537-559). Therapeutic efficacy of classicalantidepressants has been shown to be preceded by or to coincide withrestoration of the disturbed HPA axis in depression (Holsboer andBarden, 1996, supra). It has been postulated that any intervention whichcan restore this HPA dysfunction may have antidepressant potential.

One type of such intervention, studies of which support the impressionthat HPA-axis functioning and high circulating cortisol is a majorinstigator of major depression is the administration of glucocorticoidsynthesis inhibitors, as has been shown in patients suffering fromCushing's Syndrome, which is a condition in which high cortisol levelsare reported as a result of adrenal gland malfunction (due to apituitary tumour or a secondary tumour, both producing the cortisolsecretagogue ACTH). The depressive symptoms associated with Cushing'sdisappear relatively quickly with the return of cortisol levels tonormal. Such treatment may involve removal of the offending tumour ortreatment with cortisol synthesis inhibitors such as metyrapone,ketoconozole, or aminoglutethimide (Murphy, B. E. P, Steroids andDepression. J. Steroid Biochem & Mol. Biol. 38, 537-558, 1991).Similarly, relatively recent clinical trials have demonstrated thatcortisol synthesis inhibitors can be used to ameliorate depressivesymptoms in severe, treatment-resistant non-Cushing depressives (Murphy,B. E. P, Can. J. Psych. 43, 279-286, 1998; see also U.S. Pat. No.4,814,333 (Ravaris, C. L.)).

Another type of intervention is the use of direct GCR antagonists, whichhave much more specific pharmacological effects as compared to synthesisinhibitors and which may help restore HPA activity. Small scale pilotclinical studies have been conducted in order to study theantidepressant activity of the non-selective glucocorticoid receptorantagonist RU 486 (mifepristone; Murphy, B. E. P. et al. J. Psychiat.Neurosc. 18, 209-213, 1993). More recently (Nemeroff, C., RemeronScientific Expert Meeting, Budapest, Mar. 29-Apr. 1, 2001) it wasdemonstrated in a Phase IIB continuation of this study, that both thenumber of responders as well as the efficacy of the psychosis treatmentincreased with increasing daily dose of mifepristone as measured by thechange in Brief Psychiatric Rating Scale (50 mg-33% change; 600 mg-40%change and 1200 mg-52% change). These data indicate that a higher doseof glucocorticoid receptor antagonist and/or active agent is correlatedwith a higher clinical efficacy.

Non-response to standard treatments, however, reach levels as high as50%. (Connolly K R, Drugs. 2011; 71: 43-64.) Frequently, extrainterventions are necessary to get patients to achieve remission.Various augmentation and combination strategies have been described inthe literature for difficult to treat major MMD patients.

Use of an HPA-axis modulating drug in these patients has not beenstudied in spite of the fact that there is clear evidence that at leasta sub-group of MDD patients have significant HPA-axis dysfunction, asnoted above. Biological symptoms, indicative of excessive activity ofthe HPA-axis, have been reported with great consistency. In parallel,there is a body of evidence suggesting that there is an associationbetween HPA-axis functioning and treatment response, where high HPA-axisactivation at baseline, or post-treatment, is associated with a poorerresponse to S SRI treatment or a higher relapse risk.

Preclinical studies indicate that HPA-axis dysfunction of the type seenin affective disorders can attenuate the neurochemical effects of aselective serotonin re-uptake inhibitor (S SRI) antidepressant.Conversely, in animals with normal HPA axis function, co-administrationof GR antagonists augmented the neurochemical effects of an SSRI. Thesedata provide a mechanistic underpinning of the GR antagonistaugmentation strategy, and moreover indicate that the strategy may proveefficacious in patients both with and without HPA axis dysfunction.

Small scale pilot clinical studies were conducted in order to study theantidepressant activity of the non-selective glucocorticoid receptorantagonist RU 486 (mifepristone; Murphy, B. E. P. et al. J. Psychiat.Neurosc. 18, 209-213, 1993). A double blind, 4 week, paroxetinecontrolled study of PT150 (ORG 34517) in depressed patients was carriedout. Paroxetine is a selective serotonin re-uptake inhibitor which isrecognized as an effective antidepressant for major depression. Patientswere selected which had a primary depressive disorder fulfilling thediagnostic criteria of a MDD as defined by the DSM-IV for recurrent(296.3) episodes, and who had a severity of depression which resulted ina total score of at least 22 on the HAMD-21 (Hamilton Rating Scale forDepression; see Hamilton, M. “A rating scale for depression.” J. Neurol.Neurosurg. Psychiat. 1960, 23, 56-62) scale at baseline. Patients had anepisode of depression which had lasted at least 2 weeks before baseline.In this study, patients were randomly allocated to one of threetreatment groups. Group I patients (50 patients) received 2 capsuleswith 75 mg of PT150 and one placebo (total daily dose 150 mg) for thefirst 2 weeks and 2 capsules with 75 mg PT150 and 1 capsule with 150 mg(total daily dose 300 mg) the next 2 weeks; Group II patients (46patients) received 3 capsules with 150 mg PT150 (total daily dose 450mg) in the first 2 weeks and 4 capsules of PT150 (total daily dose 600mg) in the next 2 weeks; Group III patients (44 patients) received 2capsules with 10 mg paroxetine and one placebo capsule (total daily dose20 mg) for the first 2 weeks, followed by 2 capsules of 10 mg and onecapsule of 20 mg paroxetine (total daily dose 40 mg) in the next 2weeks. Medication was administered orally in the morning. Efficacyassessment was done on days 4, 7, 10, 14, 21, 28 and 35 by using the21-item HAMD scale.

Thus, GCR antagonist or active agent therapy could prove a usefulmechanism for treatment of selected individuals who fail to respond tocurrent anti-depressant therapies such as SSRIs, providing a way toenhance responsiveness or as an alternate means of achieving amaintained euthymia.

Psychotic Depression.

The present invention relates to the use of a GCR antagonist, such asfor example, ORG34517, PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof, forthe prevention or treatment of psychotic depression. Psychotic majordepression has long been recognized as a distinct psychiatric illness,having both psychotic and depressive components in a differentialdiagnosis. Psychotic major depression is very common. It has beenestimated that twenty five percent of depressed patients admitted to thehospital have psychotic major depression (Coryell (1984) J. Nerv. Ment.Dis. 172:521). Like major depression, psychotic depression is often alsoa result of high circulating cortisol levels. Various evidence supportsthis concept. Psychosis has been associated with Cushing's syndrome(Gerson (1985) Can. J. Psychiatry 30:223-224; Saad (1984) Am. J. Med.76:759-766). A GR antagonist has been used to treat acute psychiatricdisturbances secondary to Cushing's syndrome. One study showed that arelatively high dose of such a GR antagonist (400 to 800 mg per day) wasuseful in rapidly reversing acute psychosis in patients with severeCushing's' Syndrome due to adrenal cancers and ectopic secretion of ACTHfrom lung cancer (Van der Lely (1991) Ann. Intern. Med. 114:143; Van derLely (1993) Pharmacy World & Science 15:89-90; Sartor (1996) supra).Relatively high dose mifepristone, in the range of 8-12 mg/kg/day, overa relatively short period of time (4 days), was also shown to beeffective in the treatment of psychosis associated with psychotic majordepression (International Patent Application WO 99/17779; Schatzberg andBelanoff).

Surgery-Associated Immune Suppression in the Elderly.

In healthy, young to middle aged subjects suffering from stress, thereis a physiological balance between pro-inflammatory andanti-inflammatory mediators. In the elderly, the immune response isblunted as a result of the decline in several components of the immunesystem (immune senescence) and a shifting to a chronic pro-inflammatorystatus (the so-called “inflammaging” effect (Butcher and Lord, (2004)Aging Cell, pp. 151-160).

As production of cortisol remains reasonably constant with age, whereassummed levels of DHEA and DHEAS decrease gradually from the thirddecade, reaching 10-20% of their maximum by the eighth decade, Butcherand Lord (2004, supra) propose a model for age and stress, in which theage-related increase in the ratio of cortisol to DHEAS, combined with anelevated cortisol release during stress, leads to a significantreduction of immunity in aging subjects. This is proposed to explainthat aging subjects are far more prone to infections under conditions ofstress. (Butcher and Lord (2004, supra); Butcher et al. (2005, AgingCell 5, pp. 319-324).

The present invention relates to the use of a GCR antagonist, such asfor example, ORG34517, PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof, forthe prevention or treatment of infections or infectious conditions, inan aging patient, such as a human subject. The beneficial effects ofsaid GCR antagonists may be explained on the basis of their correctinginfluence on the cortisol/DHEA(S) ratio. It is believed that the effectin selected subjects, found to have high circulating cortisol by asaliva test as provided for by this invention, can be explained by theunbalanced immunosuppressive role of the increased cortisol/DHEAS ratioin the aged group in comparison to the balanced influence of cortisoland DHEAS on the immune system in normal subjects.

The meaning of the term ‘aging subject’ or ‘aged subject’ will be wellunderstood in the context of the use according to this invention.Although it is not linked to an exact lower age limit this generalnotion refers in the human situation usually to a person of at least 55years old, but it is more clear with a lowest age limit set at 60, 65,70 or 75 years.

In the context of the invention, the infection or infectious conditioncan be caused by any of several agents, e.g., by bacteria, by viruses orby fungi. Also in the context of the present invention, the expression“infectious conditions” means silent or subclinical infections as wellas conditions not resulting in a manifest infectious disease, but inwhich at least one parameter associated with an infectious disease, suchas the white blood (e.g., neutrophil, basophil or eosinophil) cellcounts or the level of some antibodies or some cytokines is higher thannormal. Normal values are known to the expert and may be found instandard medical manuals.

Particular uses according to the invention relate to aging subjectssuffering from an infection or an infectious condition concomitant tostress resulting from a trauma. The invention particularly relates touses wherein the subject suffers from the consequences of a bonefracture and/or bone surgery, either for such injury or for jointreplacement for osteoarthritis or rheumatoid arthritis. The inventionalso relates to uses wherein the subject suffers from an infection or aninfectious condition concomitant to psychological stress, particularlyacute emotional stress.

Post Traumatic Stress Disorder (PTSD).

PTSD is a severe anxiety disorder that can develop after exposure to anyevent that results in psychological trauma. This event may involve thethreat of death to oneself or to someone else, or to one's own orsomeone else's physical, sexual, or psychological integrity,overwhelming the individual's ability to cope. As an effect ofpsychological trauma, PTSD is less frequent and more enduring than themore commonly seen acute stress response. Diagnostic symptoms for PTSDinclude re-experiencing the original trauma(s) through flashbacks ornightmares, avoidance of stimuli associated with the trauma, andincreased arousal, such as difficulty falling or staying asleep, anger,and hyper-vigilance. Formal diagnostic criteria (both DSM-IV-TR andICD-9) require that the symptoms last more than one month and causesignificant impairment in social, occupational, or other important areasof functioning. (Diagnostic and statistical manual of mental disorders:DSM-IV. American Psychiatric Association. 1994. Washington, D.C.:American Psychiatric Association.) PTSD displays biochemical changes inthe brain and body that differ from other psychiatric disorders such asmajor depression. Abundant evidence suggests derangement of HPA-axisphysiology in individuals diagnosed with PTSD, though the nature of thederangements is variable: some have low cortisol, some have normallevels, others have high levels of cortisol and for some, levels may benormal, but circadian rhythm is lost. It is postulated that thesereflect different baseline mechanisms, but that when cortisol is high,either in a sustained way through the day or by loss of circadian rhythmwith elevated night time levels, it is likely to be an importantcomponent of the clinical symptomatology (Lindley S E, et al. Basal anddexamethasone suppressed salivary cortisol concentrations in a communitysample of patients with posttraumatic stress disorder. Biol. Psychiatry2004; 55: 940-5). In such patients, determined by salivary cortisoltesting, administration of a GCR antagonist is expected to betherapeutic or beneficial for the symptoms of PTSD. Prevention of WeightGain in Patients Using Anti-Psychotic and Anti-Depressant Medications.Anti-psychotic and some anti-depressant medications (e.g., SSRIs) areamongst the most important tools for treating psychiatric conditions ofall kinds. However, management of patients on who take many of thesemedications for chronic, long term disease is made difficult by theirsignificant side effect profiles. One of the most important of these isweight gain and the attendant metabolic syndrome that follows. Forexample, it is estimated that 40-80% of patients who are under chronicanti-psychotic administration experience substantial weight gain, oftenexceeding 20% or more over their ideal body weights (Umbricht et al. J.Clin. Psychiatry 1994; 55: 157-160; Khan A Y, et al. J Psychiatr Pract.2010; 16: 289-96; Pramyothin P, Khaodhiar L. Curr Opin EndocrinolDiabetes Obes. 2010; 17: 460-6; Rummel-Kluge C et al. Schizophr Res.2010; 123: 225-33). Such weight gain is one of the most common causes ofpoor compliance with anti-psychotic and anti-depressant regimens and,therefore, of long term failure of therapy. Furthermore, anti-psychoticmedications specifically are commonly associated with development ofinsulin resistance and metabolic syndrome (with development of type 2diabetes mellitus and hyper/dyslipidemia states) and the potentially andsignificantly increased risks for cardiovascular disease; theseconditions are of tremendous medical consequence for patients who arethereby caught in a “can't live with them, can't live without them”treatment scenario. While weight gain is potentially seen with allanti-psychotic medications, they are particularly common and tend tomore severe with the newer or “atypical” AP drugs (Allison et al. Am JPsychiatry 1999; 156:1686-1696; Rummel-Kluge C et al. Schizophr Res.2010; 123: 225-33).

Elevations in cortisol are associated with changes in body fat andinsulin resistance. Several years ago, in a proof of principle clinicalexperiment, it was reported that one GCR anatogonist (mifepristone) wasa highly effective treatment for multiple medical complications in apatient with Cushing's disease whose illness had not responded tosurgery and radiation, including reversal of insulin dependent diabetes:the patient was able to stop insulin within a month (Chu et al., J.Clin. Endocrinol. Metab. 2001; 86, 3568-3573.). These data suggest thata GCR antagonist could be useful for blocking and reversing the insulinresistance and weight changes seen in some patients treated withatypical antipsychotic agents. To this end, this compound was tested inrats who had olanzapine-induced weight gain and increases in abdominalfat; reversal of weight gain was seen and reduction of abdominal fat wasobtained (Beebe et al. Behay. Brain Res. 2006; 171, 225-229). A clinicaltrial with this compound then confirmed this benefit in humans with a 2week study of 600 mg/day of mifepristone that reduced olanzapine-inducedweight gain in 57 non-overweight healthy males with Body Mass Indicesless than 25 (Gross et al., Adv Ther. 2009; 26: 959-69.). Thus, GCRantagonist or active agent therapy could prove a useful mechanism totarget in treating psychotic patients with atypical antipsychoticagents. The present invention relates to the use of a GCR antagonist,such as for example, ORG34517, PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof, forthe prevention or treatment of PTSD.

Cushing's Syndrome

Cushing's Syndrome is a set of conditions in which high levels ofcirculating cortisol or other GCR agonists cause a set of seriouslydebilitating and sometimes life threatening signs and symptomsincluding, but not limited to, psychiatric disturbances (e.g. anxiety,depression, psychosis), immunosuppression, insulin resistance andmetabolic syndrome, skin conditions, hypertension and osteoporosis.Endogenous cortisol may be produced by ACTH-secreting, benign ormalignant tumors of the pituitary gland (“Cushing's Disease”) or of theadrenal cortex. These are rare conditions and therefore Cushing'sSyndrome is considered an “orphan disease.”

A proof of concept trial using RU486 to treat patients withtumor-related Cushing's Syndrome demonstrated efficacy in remittingsymptoms such as glucose metabolic abnormalities (i.e., glucoseintolerance; (group 1) and hypertension (group 2). Statisticallysignificant improvement was achieved for both groups: with 60%responding in the glucose intolerant group and 43% in the hypertensivegroup (Corcept Therapeutics Press Release Dec. 22, 2010). Thus, GCRantagonist or active agent therapy can be expected to provide clinicalbenefits for patients with Cushing's Syndrome administered prior totumor surgery to improve surgical outcomes and/or post-surgery tomitigate symptoms in patients for whom surgical cure is not achievable.

In addition, GCR antagonist or active agent therapy can be expected toprovide clinical benefits for patients, for example, in hospitals,nursing homes, nurseries, daycares, schools, work environments, publictransportation, healthcare settings, psychiatric institutions, andlong-term nursing facilities.

The present invention relates to the use of a GCR antagonist, such asfor example, ORG34517, PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof, forthe prevention or treatment of Cushing's Syndrome.

Diagnostic Systems and Kits

A diagnostic kit may comprise some or all of the followingcomponents: 1) one or more standards comprised of one or more of thebiomarker(s) of the invention, such as cortisol; 2) a ligand, such as anantibody or a plurality of antibodies, that are specific for thebiomarker(s) that are to be assayed for using the kit; 3) writteninstructions; 4) diluents for samples and the standards; 5) a washbuffer; 6) color reagents; 7) stop solution; and 8) a ligand carrier,such as an antibody carrier, for example, a lateral flow device, or amicroplate with bound antibody, or polystyrene beads. An example of sucha kit is a quantitative ELISA (enzyme-linked immunosorbent assay) thatdetermines the concentration or concentrations of the biomarker orbiomarker(s) in accordance with methods embodied by the invention. Theprinciple of the assay is to use the quantitative sandwich enzymeimmunoassay technique wherein a monoclonal or polyclonal antibodyselective for a biomarker is pre-coated onto a carrier such as amicroplate into its wells. The standards and sample are then pipettedinto the wells and any of the biomarker that is present is bound to thisimmobilized antibody. Next, the wells are washed with washing buffer,and an enzyme-linked monoclonal or polyclonal antibody that is specificfor the biomarker is added to the wells.

Washing is again performed, then a substrate solution is added to thewells. Color subsequently develops in proportion to the amount ofpolypeptide of the invention that is bound in the first step. The colordevelopment is stopped using a stop solution, and the intensity of thecolor is measured by a microplate reader.

The methods of the invention may be carried out using, for example, alateral flow assay. Such lateral flow assays have the potential to be acost-effective, fast, simple, and sensitive method, for instance foron-site screening assays. The lateral flow assay comprises a carrierthat allows a lateral flow to occur wherein either the sample or thedetection reagent is displaced form one location on the carrier toanother. There are many formats of lateral flow assays suitable for usein a method embodied by the invention, and the skilled person willreadily know how to select and optimize a particular format. An exampleof a lateral flow test strip of the invention comprises, for example,the following components:

Sample pad—an absorbent pad onto which the test sample is applied.Conjugate or reagent pad—this contains antibodies specific to the targetanalyte conjugated to colored particles (usually colloidal goldparticles, or latex microspheres).Reaction membrane—typically a hydrophobic nitrocellulose or celluloseacetate membrane onto which antitarget analyte antibodies areimmobilized in a line across the membrane as a capture zone or test line(a control zone may also be present, containing antibodies specific forthe conjugate antibodies).Wick or waste reservoir—a further absorbent pad designed to draw thesample across the reaction membrane by capillary action and collect it.

Double Antibody Sandwich Assays—

In this format the sample migrates from the sample pad through theconjugate pad where any target analyte present will bind to theconjugate. The sample then continues to migrate across the membraneuntil it reaches the capture zone where the target/conjugate complexwill bind to the immobilized antibodies producing a visible line on themembrane. The sample then migrates further along the strip until itreaches the control zone, where excess conjugate will bind and produce asecond visible line on the membrane. This control line indicates thatthe sample has migrated across the membrane as intended. Two clear lineson the membrane is a positive result.A single line in the control zone is a negative result. Double antibodysandwich assays are most suitable for larger analytes, such as bacterialpathogens and viruses, with multiple antigenic sites. Competitive assaysare primarily used for testing small molecules and differ from thedouble antibody sandwich format in that the conjugate pad containsantibodies that are already bound to the target analyte, or to ananalogue of it. If the target analyte is present in the sample it willtherefore not bind with the conjugate and will remain unlabelled.

As the sample migrates along the membrane and reaches the capture zone,an excess of unlabelled analyte will bind to the immobilized antibodiesand block the capture of the conjugate, so that no visible line isproduced.

The unbound conjugate will then bind to the antibodies in the controlzone producing a visible control line. A single control line on themembrane is a positive result. Two visible lines in the capture andcontrol zones is a negative result. However, if an excess of unlabelledtarget analyte is not present, a weak line may be produced in thecapture zone, indicating an inconclusive result. Competitive assays aremost suitable for testing for small molecules, such as mycotoxins,unable to bind to more than one antibody simultaneously. There are anumber of variations on lateral flow technology. The capture zone on themembrane may contain immobilized antigens or enzymes-depending on thetarget analyte—rather than antibodies. It is also possible to applymultiple capture zones to create a multiplex test. For example,commercial test strips able to detect both EHEC Shiga toxins ST1 and ST2separately in the same sample have been developed. Lateral flowimmunoassays are simple to use by untrained operators and generallyproduce a result within 15 minutes. They are very stable and robust,have a long shelf life and do not usually require refrigeration. Theyare also relatively inexpensive to produce. These features make themideal for use at the point-of-care and for testing samples in the field,as well as in the laboratory. However, their sensitivity is limitedwithout additional concentration or culture procedures.

Quantitative Tests—

While most lateral flow immunoassays are only capable of providing aqualitative result, it is possible to obtain some degree ofquantification by measuring the amount of conjugate bound to the capturezone. This can be done using a dedicated reader to measure the intensityof the colored test line. For example, the Neogen Corporation hasdeveloped the Accuscan™ lateral flow reader for use with its range ofReveal® assay kits and Charm Sciences also supplies a reader for itsRosa® range of mycotoxin test strips. More sophisticated techniques,such as fluorescent dye labeled conjugates, have also been developed toimprove the quantitative potential of lateral flow assays. Applicationsin the 20 years since the first lateral flow test was launched haveexpanded to include a huge range of different tests that have beendeveloped based on the same technology. The first commercially availablekits were aimed at the clinical diagnostics field, but there are nowproducts with applications in almost every branch of microbiology.Clinical microbiology—lateral flow tests have been developed forbacterial pathogens, respiratory and enteric viruses, intestinalparasites and bacterial toxins. Many of the lateral flow immunoassayproducts designed for the clinical sector were intended to be used atthe point-of-care for direct testing of fecal, blood and urine samplesand nose and throat swabs, where the simple operation and speed of thetests is key to their use outside of the laboratory. However, the sametest strips may also be useful as a quick confirmatory test followinglaboratory culture of clinical samples. Food and agriculturalmicrobiology—test strips are available for food borne bacterialpathogens, bacterial and fungal toxins. In the food microbiology sector,the main applications are more likely to be in the laboratory, althoughthere are field test kits for mycotoxins in grain samples. Testing forfood borne bacterial pathogens generally involves at least oneenrichment stage before the assay strip is used to confirm the presenceor absence of the pathogen. Some manufacturers, such as Dupont®, havedeveloped enrichment media and methods specifically designed for usewith lateral flow test strips. Test strips may also be useful for rapidconfirmation of the identity of bacterial isolates from conventionalmicrobiological testing.

A diagnostic system in kit form of the present invention includes, forexample, in an amount sufficient for at least one assay, a polypeptide,antibody composition or monoclonal antibody composition of the presentinvention, as a packaged reagent. Instructions for use of the packagedreagent are also typically included.

A diagnostic system in kit form of the present invention may include,for example, a means for detecting the presence of a biologicalsubstance in a test sample, comprising for example, a lollipop-likeapparatus including a stem integrated with the base and a headintegrated with the stem, for collecting a test sample consisting of,for example, saliva, or a bodily fluid sample from a subject. The stemhead may include a receptor of a sponge like carrier to ensure a highvoid volume to absorb sufficient saliva, or bodily fluid sample. SeeU.S. Pat. No. 7,993,283, incorporated by reference herein in itsentirety.

A diagnostic system in kit form of the present invention may include,for example, a means for combining the test sample with a bufferingsystem (Reagent 1) containing viscosity controllers and stabilizers intoa reaction vessel and mixing the solution. A diagnostic system in kitform of the present invention may include, for example, a means forreading the a parameter of the reaction vessel with sample and buffer,and further means for combining the test sample and buffer mixture witha fluorescence-labeled ligand (Reagent 2) to said biological substancein the reaction vessel, mixing the solution to produce an assaysolution. Furthermore,

Reagent 2 may be delivered to the reaction vessel without furtherdilution volume of the assay solution.

As used herein, the term “package” refers to a solid matrix or materialsuch as glass, plastic, paper, foil and the like capable of holdingwithin fixed limits a polypeptide, antibody composition or monoclonalantibody composition of the present invention. Thus, for example, apackage can be a glass vial used to contain milligram quantities of acontemplated polypeptide or it can be a microtiter plate well to whichmicrogram quantities of a contemplated polypeptide have been operativelyaffixed, i.e., linked so as to be capable of being immunologically boundby an antibody. “Instructions for use” typically include a tangibleexpression describing the reagent concentration or at least one assaymethod parameter such as the relative amounts of reagent and sample tobe admixed, maintenance time periods for reagent/sample admixtures,temperature, buffer conditions and the like.

In preferred embodiments, a diagnostic system of the present inventionfurther includes a label or indicating means capable of signaling theformation of a complex containing a polypeptide or antibody molecule ofthe present invention.

The word “complex” as used herein refers to the product of a specificbinding reaction such as an antibody-antigen or receptor-ligandreaction. Exemplary complexes are immunoreaction products.

As used herein, the terms “label” and “indicating means” in theirvarious grammatical forms refer to single atoms and molecules that areeither directly or indirectly involved in the production of a detectablesignal to indicate the presence of a complex. Any label or indicatingmeans can be linked to or incorporated in an expressed protein,polypeptide, or antibody molecule that is part of an antibody ormonoclonal antibody composition of the present invention, or usedseparately, and those atoms or molecules can be used alone or inconjunction with additional reagents such labels are themselveswell-known in clinical diagnostic chemistry and constitute a part ofthis invention only insofar as they are utilized with otherwise novelproteins methods and/or systems.

The labeling means can be a fluorescent labeling agent that chemicallybinds to antibodies or antigens without denaturing them to form afluorochrome (dye) that is a useful immunofluorescent tracer. Suitablefluorescent labeling agents are fluorochromes such as fluoresceinisocyanate (FIC), fluorescein isothiocyante (FITC),5-dimethylamine-1-naphthalenesulfonyl chloride (DANSC),tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200sulphonyl chloride (RB 200 SC) and the like. A description ofimmunofluorescence analysis techniques is found in DeLuca,“Immunofluorescence Analysis”, in Antibody As a Tool, Marchalonis, etal., eds., John Wiley & Sons, Ltd., pp. 189-231 (1982), which isincorporated herein by reference.

In preferred embodiments, the indicating group is an enzyme, such ashorseradish peroxidase (HRP), glucose oxidase, or the like. In suchcases where the principal indicating group is an enzyme such as HRP orglucose oxidase, additional reagents are required to visualize the factthat a receptor-ligand complex (immunoreactant) has formed. Suchadditional reagents for HRP include hydrogen peroxide and an oxidationdye precursor such as diaminobenzidine. An additional reagent usefulwith glucose oxidase is 2,2′-azino-di-β-ethyl-benzthiazoline-G-sulfonicacid) (ABTS).

Radioactive elements are also useful labeling agents and are usedillustratively herein. An exemplary radiolabeling agent is a radioactiveelement that produces gamma ray emissions. Elements which themselvesemit gamma rays, such as .sup. 1241, .sup. 1251, .sup. 1281, .sup. 1321and, sup.51Cr represent one class of gamma ray emission-producingradioactive element indicating groups. Particularly preferred is .sup.1251. Another group of useful labeling means are those elements such as.sup.llC, sup, 18F, .sup.150 and, sup, 13N which themselves emitpositrons. The positrons so emitted produce gamma rays upon encounterswith electrons present in the animal's body. Also useful is a betaemitter, such .sup.lllindium or, sup.3H.

The linking of labels, i.e., labeling of, polypeptides and proteins iswell known in the art. For instance, antibody molecules produced by ahybridoma can be labeled by metabolic incorporation ofradioisotope-containing amino acids provided as a component in theculture medium. See, for example, Galfire et al., Meth. Enzymol.,73:3-46 (1981). The techniques of protein conjugation or couplingthrough activated functional groups are particularly applicable. See,for example, Aurameas, et al., Scand. J. Immunol., Vol. 8 Suppl. 7:7-23(1978), Rodwell et al., Biotech., 3:889-894 (1984), and U.S. Pat. No.4,493,795, which are all incorporated herein by reference. Thediagnostic systems can also include, preferably as a separate package, aspecific binding agent. A “specific binding agent” is a molecular entitycapable of selectively binding a reagent species of the presentinvention or a complex containing such a species, but is not itself apolypeptide or antibody molecule composition of the present invention.Exemplary specific binding agents are second antibody molecules,complement proteins or fragments thereof, S. aureus protein A, and thelike. Preferably the specific binding agent binds the reagent specieswhen that species is present as part of a complex.

In preferred embodiments, the specific binding agent is labeled.However, when the diagnostic system includes a specific binding agentthat is not labeled, the agent is typically used as an amplifying meansor reagent. In these embodiments, the labeled specific binding agent iscapable of specifically binding the amplifying means when the amplifyingmeans is bound to a reagent species-containing complex.

The diagnostic kits of the present invention can be used in an “ELISA”format to detect, for example, the presence or quantity of cortisol in abody fluid sample such as serum, plasma, or urine, etc. “ELISA” refersto an enzyme-linked immunosorbent assay that employs an antibody orantigen bound to a solid phase and an enzyme-antigen or enzyme-antibodyconjugate to detect and quantify the amount of an antigen or antibodypresent in a sample. A description of the ELISA technique is found inChapter 22 of the 4th Edition of Basic and Clinical Immunology by D. P.Sites et al., published by Lange Medical Publications of Los Altos,Calif., in 1982 and in U.S. Pat. Nos. 3,654,090; 3,850,752; and4,016,043, which are all incorporated herein by reference.

Thus, for example, a polypeptide, antibody molecule composition ormonoclonal antibody molecule composition of the present invention can beaffixed to a solid matrix to form a solid support that comprises apackage in the subject diagnostic systems. The reagent is typicallyaffixed to the solid matrix by adsorption from an aqueous mediumalthough other modes of affixation, well known to those skilled in theart, can be used.

Useful solid matrices are also well known in the art. Such materials arewater insoluble and include cross-linked dextran; agarose; beads ofpolystyrene beads about 1 micron to about 5 millimeters in diameter;polyvinyl chloride, polystyrene, cross-linked polyacrylamide,nitrocellulose- or nylon-based webs such as sheets, strips or paddles;or tubes, plates or the wells of a microtiter plate such as those madefrom polystyrene or polyvinylchloride.

The reagent species, labeled specific binding agent or amplifyingreagent of any diagnostic system described herein can be provided insolution, as a liquid dispersion or as a substantially dry power, e.g.,in lyophilized form. Where the indicating means is an enzyme, theenzyme's substrate can also be provided in a separate package of asystem. A solid support such as the before-described microtiter plateand one or more buffers can also be included as separately packagedelements in this diagnostic assay system.

The packaging materials discussed herein in relation to diagnosticsystems are those customarily utilized in diagnostic systems. Suchmaterials include glass and plastic (e.g., polyethylene, polypropyleneand polycarbonate) bottles, vials, plastic and plastic-foil laminatedenvelopes and the like. In one embodiment a diagnostic system of thepresent invention is useful for assaying for the presence of, forexample, cortisol. Such a system comprises, in kit form, a packagecontaining an antibody to, for example, cortisol.

“Sample” refers to, for example, essentially any source from whichmaterials of interest to be analyzed (e.g., ligands and antiligands,such as antibodies and antigens, and nucleic acids and theircomplements) can be obtained. A sample may be acquired from essentiallyany organism, including animals and plants, as well as cell cultures,recombinant cells and cell components. Samples can be from a biologicaltissue, fluid or specimen and may be obtained from a diseased or healthyorganism. Samples may include, but are not limited to, saliva, sputum,amniotic fluid, blood, blood cells (e.g., white cells), urine, semen,peritoneal fluid, pleural fluid, tissue or fine needle biopsy samples,and tissue homogenates. Samples may also include sections of tissuessuch as frozen sections taken for histological purposes. Typically,samples are taken from a human. However, samples can be obtained fromother mammals also, including by way of example and not limitation,dogs, cats, sheep, cattle, and pigs. The sample may be pretreated asnecessary by dilution in an appropriate buffer solution or concentrated,if desired. Any of a number of standard aqueous buffer solutions,employing one of a variety of buffers, such as phosphate, Tris, or thelike, preferably at physiological pH can be used.

Biological samples can be derived from patients using well knowntechniques such as venipuncture, lumbar puncture, fluid sample such assaliva or urine, or tissue biopsy and the like. When the biologicalmaterial is derived from non-humans, such as commercially relevantlivestock, blood and tissue samples are conveniently obtained fromlivestock processing plants. Alternatively, a biological sample may beobtained from a cell or blood bank where tissue and/or blood are stored,or from an in vitro source, such as a culture of cells.

Techniques for establishing a culture of cells for use as a source forbiological materials are well known to those of skill in the art.

In one embodiment the sample is selected from or is derived from, forexample, microbial products or biological products.

Although the above described example relates to the antigens relating todisease, the immunoassay apparatus could be used, for example, as anallergy test kit, as a test kit for drugs of abuse or for analyzingnon-human derived samples e.g. bovine, porcine, and veterinary tests.Specific reagents used in the assay device will be selected so as toensure that the particular target analyte is detected as is well knownin the art. The target analyte may be any analyte for example a chemicalreagent which may be organic or inorganic, and which optionallycomprises a hapten, a protein, a polypeptide, a microorganism or anucleic acid sequence.

In particular, the analyte is a hormone such as a fertility hormone likeprogesterone or a stress hormone such as cortisol. However, there is awide range of applications of these types of tests across the entirefield of diagnostics and analysis. Detection of marker proteins orhormones can be diagnostic of certain disease conditions in humans oranimals, and the presence of drugs or drug residues may also be requiredto be detected, for example, in animal husbandry, forensic medicine orin the testing for banned or prohibited drug substances.

Alternatively, the analyte is a chemical reagent, for instance a smallmolecule, which suitably comprises a hapten. Small molecules willgenerally comprise a single recognizable binding site. Typically theywill have a molecular weight of less than 1 kDa.

Where the assay utilizes a labelled binding partner for the analyte andthe analyte is a chemical reagent, the binding partner may comprise anyother reagent which reacts with or otherwise becomes associated with thechemical reagent, either because it forms covalent or ionic bonds withthe reagent, or by the formation of other interactions, such as hydrogenbonding or Van der Waals interactions. For example, where the chemicalreagent is an acid, the binding partner may comprise an alcohol or anamine that forms an ester or amide with the acid under the sorts ofconditions found in the test. Alternatively the binding partner maycomprise a base that forms a salt with the acid. Conversely, where thebinding partner may comprise the acid part of the reactive pair.

Where the analyte is or comprises a hapten or a protein antigen, thebinding partner may comprise an antibody or a binding fragment thereof,which may be monoclonal, polyclonal or recombinant, but preferably ismonoclonal. Where the analyte is a hormone or enzyme, the labelledbinding partner may comprise a labelled receptor for the analyte.However, where the analyte is itself an immunoglobulin, and inparticular, an antibody, the labelled binding partner may also comprisefor instance, an antigen or recombinant antigen, as well as antiantibodyimmunoglobulin such as anti-sera.

Antibodies or binding fragments to small molecules such as haptens, aregenerated by attaching the molecule to an immunogenic reagent andadministering this to an animal such as a mouse or rabbit. Antibodiesare then harvested from the animal in the usual way. Monoclonalantibodies are obtained by fusing spleen cells to hybridoma cells, andselecting those which bind the hapten, using routine procedures.

For example, where the analyte is a biologically active material such asan active agrochemical as discussed above, specific reagents used in theassay device will be selected so as to ensure that the particular targetbiologically active material is detected as is well known in the art.The biologically active material may be any active chemical such as anagrochemical, for example a chemical reagent which may be organic orinorganic, and which optionally comprises a hapten, a protein, apolypeptide, a microorganism or a nucleic acid sequence. Most preferablythe biologically active material is a chemical reagent, for instance asmall molecule, which suitably comprises a hapten. Small molecules willgenerally have a single antibody binding site. Typically they will havea molecular weight of less that 1 kDa.

Antibodies or binding fragments to small molecules such as haptens, aregenerated by attaching the molecule to an immunogenic reagent andadministering this to an animal such as a mouse or rabbit. Antibodiesare then harvested from the animal in the usual way. Monoclonalantibodies are obtained by fusing spleen cells to hybridoma cells, andselecting those which bind the hapten, using routine procedures.

Microarrays

The method of the invention is particularly useful in combination withthe analysis of gene expression profiles. In some embodiments, a geneexpression profile, such as a collection of transcription rates of anumber of genes, is converted to a projected gene expression profile.The projected gene expression profile is a collection of expressionvalues. The conversion is achieved, in some embodiments, by averagingthe transcription rate of the genes. In some other embodiments, otherlinear projection processes may be used.

Microarrays may be prepared and analyzed using methods known in the art.Oligonucleotides may be used as either probes or targets in amicroarray. The microarray can be used to monitor the expression levelof large numbers of genes simultaneously and to identify geneticvariants, mutations, and single nucleotide polymorphisms. Suchinformation may be used to determine gene function; to understand thegenetic basis of a condition, disease, or disorder; to diagnose acondition, disease, or disorder; and to develop and monitor theactivities of therapeutic agents. (See, e.g., Brennan et al. (1995) U.S.Pat. No. 5,474,796; Schena et al. (1996) Proc. Natd. Acad. Sci.93:10614-10619; Baldeschweiler et al. (1995) PCT applicationWO95/251116; Shalon et al. (1995) PCT application WO95/35505; Heller etal. (1997) Proc. Natl. Acad. Sci. 94:2150-2155; and Heller et al.(1997); U.S. Pat. No. 5,605,662.) Hybridization probes are also usefulin mapping the naturally occurring genomic sequence. The sequences maybe mapped to a particular chromosome, to a specific region of achromosome, or to artificial chromosome constructions, e.g., humanartificial chromosomes (HACs), yeast artificial chromosomes (YACs),bacterial artificial chromosomes (BACs), bacterial PI constructions, orsingle chromosome DNA libraries.

Using the methods of the invention a skilled artisan can readily selectand prepare probes for a microarray wherein the microarray containsspecific individual probes for less than all the genes in the genome andless than all the genes in the genome. In such embodiments, themicroarray contains one or two or more individual probes, each of whichhybridizes to an expression product (e.g., mRNA, or cDNA or cRNA derivedtherefrom) for a desired number of genes. Thus, for example, changes inthe expression of all or most of the genes in the entire genome of acell or organism can thereby be monitored by use of a surrogate and on asingle microarray by measuring expression of the group of genes that arerepresentative of all or most of the genes of the genome. Suchmicroarrays can be prepared using the selected probes and are thereforepart of the present invention.

Stroke

Stroke (also referred to herein as acute stroke, ischemic stroke and/orcerebrovascular ischemia) is often cited as the third most common causeof death in the industrial world, ranking behind ischemic heart diseaseand cancer. Strokes are responsible for about 300,000 deaths annually inthe United States and are a leading cause of hospital admissions andlong-term disabilities. Accordingly, the socioeconomic impact of strokeand its attendant burden on society is practically immeasurable.

“Stroke” is defined by the World Health Organization as a rapidlydeveloping clinical sign of focal or global disturbance of cerebralfunction with symptoms lasting at least 24 hours. Strokes are alsoimplicated in deaths where there is no apparent cause other than aneffect of vascular origin. Strokes are typically caused by blockages orocclusions of the blood vessels to the brain or within the brain.

With complete occlusion, arrest of cerebral circulation causes cessationof neuronal electrical activity within seconds. Within a few minutesafter the deterioration of the energy state and ion homeostasis,depletion of high energy phosphates, membrane ion pump failure, effluxof cellular potassium, influx of sodium chloride and water, and membranedepolarization occur. If the occlusion persists for more than five toten minutes, irreversible damage results. With incomplete ischemia,however, the outcome is difficult to evaluate and depends largely onresidual perfusion and the availability of oxygen. After a thromboticocclusion of a cerebral vessel, ischemia is rarely total. Some residualperfusion usually persists in the ischemic area, depending on collateralblood flow and local perfusion pressure.

A subject having a stroke is so diagnosed by symptoms experienced and/orby a physical examination including interventional andnon-interventional diagnostic tools such as CT and MR imaging. Themethods of the invention are advantageous for the treatment of variousclinical presentations of stroke subjects. A subject having a stroke maypresent with one or more of the following symptoms: paralysis, weakness,decreased sensation and/or vision, numbness, tingling, aphasia (e.g.,inability to speak or slurred speech, difficulty reading or writing),agnosia (i.e., inability to recognize or identify sensory stimuli), lossof memory, co-ordination difficulties, lethargy, sleepiness orunconsciousness, lack of bladder or bowel control and cognitive decline(e.g., dementia, limited attention span, inability to concentrate).Using medical imaging techniques, it may be possible to identify asubject having a stroke as one having an infarct or one havinghemorrhage in the brain.

The present invention relates to the use of a GCR antagonist, such asfor example, ORG34517, PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof, forthe prevention or treatment of stroke.

The treatment and/or prevention of infection after stroke can be forpatients who have experienced a stroke or can be a prophylactictreatment. Short term prophylactic treatment is indicated for subjectshaving surgical or diagnostic procedures which risk release of emboli,lowering of blood pressure or decrease in blood flow to the brain, toreduce the injury due to any ischemic event that occurs as a consequenceof the procedure. Longer term or chronic prophylactic treatment isindicated for subjects having cardiac conditions that may lead todecreased blood flow to the brain, or conditions directly affectingbrain vasculature. If prophylactic, then the treatment is for subjectshaving an abnormally elevated risk of an ischemic stroke, as describedabove. If the subject has experienced a stroke, then the treatment caninclude acute treatment. Acute treatment for prevention of infectionafter stroke in a patient means administration of an agent of theinvention at the onset of symptoms of the condition or within 48 hoursof the onset, preferably within 24 hours, more preferably within 12hours, more preferably within 6 hours, and even more preferably within 3hours of the onset of symptoms of the condition.

An important embodiment of the invention is treatment of a subject withan abnormally elevated risk of an ischemic stroke. As used herein,subjects having an abnormally elevated risk of an ischemic stroke is acategory determined according to conventional medical practice; suchsubjects may also be identified in conventional medical practice ashaving known risk factors for stroke or having increased risk ofcerebrovascular events. Subjects having an abnormally elevated risk ofan ischemic stroke includes, for example, individuals undergoingsurgical or diagnostic procedures which risk release of emboli, loweringof blood pressure or decrease in blood flow to the brain, such ascarotid endarterectomy, brain angiography, neurosurgical procedures inwhich blood vessels are compressed or occluded, cardiac catheterization,angioplasty, including balloon angioplasty, coronary by-pass surgery, orsimilar procedures.

CNS Injury

Conditions suitable for treatment according to this invention include,for example, seizure disorders, pain syndromes, neurodegenerativediseases (including motor-neuron diseases, myelopathies,radiculopathies, and disorders of the sympathetic nervous system),dementias, cerebrovascular conditions, movement disorders, brain trauma,cranial nerve disorders, neuropsychiatric disorders, and other diseaseneuropathies (including viral associated neuropathies, diabetesassociated neuropathies, Guillian-Barre syndrome, dysproteinemias,transthyretin-induced neuropathies, and carpal tunnel syndrome). Thepresent invention relates to the use of a GCR antagonist, such as forexample, ORG34517, PT150, PT155, PT156, PT157, PT158, TCY1, combinationsthereof, and pharmaceutically acceptable salts thereof, for theprevention or treatment of CNS Injury.

As used herein, seizure disorders include complex partial seizures,simple partial seizures, partial seizures with secondary generalization,generalized seizures (including absence, grand mal (tonic clonic),status epilepticus, tonic, atonic, myoclonic), neonatal and infantilespasms, drug-induced seizures, trauma-induced seizures, and febrileseizures, and additional specific epilepsy syndromes such as juvenilemyoclonic epilepsy, Lennox-Gastaut, mesial temporal lobe epilepsy,nocturnal frontal lobe epilepsy, progressive epilepsy with mentalretardation, and progressive myoclonic epilepsy, as well as seizuresassociated with CNS mass lesions.

Pain syndromes include, for example, headaches (e.g., migraine, tension,and cluster), acute pain, chronic pain, neuropathic pain, nociceptivepain, central pain and inflammatory pain, drug-induced neuropathic pain,causalgia, complex regional pain syndrome types I and II, and reflexsympathetic dystrophy (RSDS).

Neurodegenerative diseases include Alzheimer's disease, Parkinson'sDisease, multiple sclerosis, Huntington's Disease, ALS, spinal muscularatrophy, muscular dystrophies prion-related diseases, cerebellar ataxia,Friedrich's ataxia, SCA, Wilson's disease, RP, Gullian Bane syndrome,Adrenoleukodystrophy, Menke's Sx, cerebral autosomal dominantarteriopathy with subcortical infarcts (CADASIL), Charcot Marie Toothdiseases, neurofibromatosis, von-Hippel Lindau, Fragile X, spasticparaplegia, tuberous sclerosis complex, Wardenburg syndrome, spinalmotor atrophies, Tay-Sach's, Sandoff disease, familial spasticparaplegia, myelopathies, radiculopathies, encephalopathies associatedwith trauma, radiation, drugs and infection, and disorders of thesympathetic nervous system (e.g., Shy Drager (familial dysautonomia),diabetic neuropathy, drug-induced and alcoholic neuropathy).

Dementias include Alzheimer's disease, Parkinson's disease, Pick'sdisease, fronto-temporal dementia, vascular dementia, normal pressurehydrocephalus, Huntington's disease, and MCI. Cerebrovascular conditionsamenable to treatment according to the present invention includecerebrovascular disease and strokes (e.g., thrombotic, embolic,thromboembolic, hemorrhagic [including AVM and berry aneurysms],venoconstrictive, and venous).

Included in movement disorders are Parkinson's disease, dystonias,benign essential tremor, tardive dystonia, tardive dyskinesia, andTourette's syndrome.

Brain trauma as used herein includes traumatic brain and spinal cordinjuries as well as brain injuries from radiation.

Cranial nerve disorders include trigeminal neuropathy, trigeminalneuralgia, Menier's syndrome, glossopharangela neuralgia, dysphagia,dysphonia, cranial nerve palsies and Bell's palsy. Neuropsychiatricdisorders include panic syndrome, general anxiety disorder, phobicsyndromes of all types, mania, manic depressive illness, hypomania,unipolar depression, depression, stress disorders, PTSD, somatoformdisorders, personality disorders, psychosis, and schizophrenia), anddrug dependence/addiction (e.g., alcohol, psychostimulants (e.g., crack,cocaine, speed, meth), opioids, and nicotine), and drug-inducedpsychiatric disorders.

Other disease neuropathies that may be treated with the compositions andmethods described herein include Guillian-Barre, diabetes associatedneuropathies, dysproteinemias, transthyretin-induced neuropathies,neuropathy associated with HIV, herpes viruses (including herpes zoster)or other viral infection, neuropathy associated with Lyme disease,carpal tunnel syndrome, tarsal tunnel syndrome, amyloid-inducedneuropathies, leprous neuropathy, Bell's palsy, compressionneuropathies, sarcoidosis-induced neuropathy, polyneuritis cranialis,heavy metal induced neuropathy, transition metal-induced neuropathy,drug-induced neuropathy, postmeningitis syndrome, post-polio syndrome,prion diseases, and radiation associated neuropathic syndromes. Otherdiseases amenable to treatment with the present invention includefatigue syndromes (e.g., chronic fatigue syndrome and fibromyalgia),ataxic syndromes, olivopontoicerebellar degeneration, striatonigraldegeneration, and axonic brain damage.

The present invention is particularly useful in the treatment ofneuropsychiatric disorders such as depression, agitation, anxiety,seizure disorders such as grand mal seizures, status epilepticus,migraine pain treatment and prophylaxis, Alzheimer's disease,Parkinson's disease, and traumatic brain and spinal cord injury.

Also, the higher doses enabled by the present invention are expected tobe of particular importance for dementias including Alzheimer's disease,Parkinson's disease, and vascular dementia, pain syndromes, includingheadaches and migraines, seizure disorders, movement disorders, andbrain trauma.

Furthermore, the ease of use and convenience of a dosage form provideddeveloped to be delivered at once per day or less frequentadministration at a therapeutically effective quantity from the onset oftherapy is of value in the treatment of dementias including Alzheimer'sdisease and Parkinson's disease, seizure disorders, pain syndromes, andcerebrovascular conditions.

Formulations for Alternate Specific Routes of Administration

The pharmaceutical compositions may be optimized for particular types ofdelivery. For example, pharmaceutical compositions for oral delivery areformulated using pharmaceutically acceptable carriers that are wellknown in the art. The carriers enable the agents in the composition tobe formulated, for example, as a tablet, pill, capsule, solution,suspension, sustained release formulation; powder, liquid or gel fororal ingestion by the subject.

The GCR antagonist may also be delivered in an aerosol spray preparationfrom a pressurized pack, a nebulizer or from a dry powder inhaler.Suitable propellants that can be used in a nebulizer include, forexample, dichlorodifluoro-methane, trichlorofluoromethane,dichlorotetrafluoroethane and carbon dioxide. The dosage can bedetermined by providing a valve to deliver a regulated amount of thecompound in the case of a pressurized aerosol.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as set outabove. Preferably the compositions are administered by the oral,intranasal or respiratory route for local or systemic effect.Compositions in preferably sterile pharmaceutically acceptable solventsmay be nebulized by use of inert gases. Nebulized solutions may bebreathed directly from the nebulizing device or the nebulizing devicemay be attached to a face mask, tent or intermittent positive pressurebreathing machine. Solution, suspension or powder compositions may beadministered, preferably orally or nasally, from devices that deliverthe formulation in an appropriate manner.

Regenerative Therapy

The activities of GR agonists and their alteration of cellular functionsare variable, depending on complex intracellular molecular signalingthat are cell and tissue specific. Amongst the cells that haveglucocorticoid receptors are stem and progenitor cells of all tissuesand organs of the body. Thus, binding of such molecules to normative,“in-tissue” stem cells and the progeny of these stem cells, so-called“transit amplifying” progenitor cells, results in variable, cell andtissue specific effects, inhibitory or enhancing of stem and progenitorcell functions, including activation, proliferation, migration anddifferentiation all of which are dependent on the tissue/organ inquestion.

The present invention relates to the use of a GCR antagonist or activeagent, such as for example, ORG34517, PT150, PT155, PT156, PT157, PT158,TCY1, combinations thereof, and pharmaceutically acceptable saltsthereof, for the prevention or treatment of, will thus block the effectsof GR-agonists in tissue specific fashion, enhancing stem/progenitorcell functioning in some, inhibiting it in others. GR-antagonists willhave beneficial effects in specific clinical settings where regenerativemedicine approaches to disease and wound healing may be of use,including: enhanced post-transplant functioning of autologous stem celltransplants (dependent on tissue of origin and/or target tissue).Attenuation of the peri-surgical effects of catabolic stress hormonesrelated to surgical or other physical traumas (e.g. combat wounds).

Systemic GR blockade will be inappropriate, but direct application ofThe present invention relates to the use of a GCR antagonist, such asfor example, ORG34517, PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof, forthe prevention or treatment of to site of injury/wounding, eithertopically (for example, to prevent wound dehiscence) or by directinjection or intravascular infusion (for visceral organ injuries) willbe beneficial.

The present invention relates to the use of cortisol blockers(glucocorticoid receptor [GR] antagonists) to treat stem cells forregenerative therapy.

Stem Cells

The term “stem cell” generally refers to a cell that on division facestwo developmental options: the daughter cells can be identical to theoriginal cell (self-renewal) or they may be the progenitors of morespecialized cell types (differentiation). The stem cell is thereforecapable of adopting one or other pathway (a further pathway exists inwhich one of each cell type can be formed). Stem cells are thereforecells which are not terminally differentiated and are able to producecells of other types.

Embryonic Stem (ESCs) cells may be isolated from the inner cell mass(ICM) of the blastocyst, which is the stage of embryonic developmentwhen implantation occurs. Pluripotent stem cells are true stem cells,with the potential to make any differentiated cell in the body. However,they cannot contribute to making the extraembryonic membranes which arederived from the trophoblast.

Several types of pluripotent stem cells have been found.

Multipotent stem cells are true stem cells but can only differentiateinto a limited number of types. For example, the bone marrow containsmultipotent stem cells that give rise to all the cells of the blood butnot to other types of cells. Multipotent stem cells are found in adultanimals. It is thought that every organ in the body contains them wherethey can replace dead or damaged cells. Methods of characterizing stemcells are known in the art, and include the use of standard assaymethods such as clonal assay, flow cytometry, long-term culture andmolecular biological techniques e.g. PCR, RT-PCR and Southern blotting.

Adult stem cells comprise a wide variety of types including neuronal,skin and the blood forming stem cells which are the active component inbone marrow transplantation. These latter stem cell types are also theprincipal feature of umbilical cord-derived stem cells. Adult stem cellscan mature both in the laboratory and in the body into functional, morespecialised cell types although the exact number of cell types islimited by the type of stem cell chosen.

Induced pluripotent stem cells, commonly abbreviated as iPS cells oriPSCs, are a type of pluripotent stem cell artificially derived from anon-pluripotent cell, typically an adult somatic cell, by insertingcertain genes. iPS cells are reviewed and discussed in Takahashi, K. &Yamanaka (2006), Yamanaka S, et. al. (2007), Wemig M, et. al. (2007),Maherali N, et. al. (2007), Yu J, et al. (2007) and Takahashi et al.,(2007), all of which are incorporated herein by reference.

iPS cells are typically derived by transfection of certain stemcell-associated genes into non-pluripotent cells, such as adultfibroblasts. Transfection is typically achieved through viral vectors,for example through retroviral reprogramming. Transfected genes includethe master transcriptional regulators Oct-3/4 (Pouf51) and Sox2,although it is suggested that other genes enhance the efficiency ofinduction. After 3-4 weeks, small numbers of transfected cells begin tobecome morphologically and biochemically similar to pluripotent stemcells, and are typically isolated through morphological selection,doubling time, or through a reporter gene and antibiotic infection.IPSCs may be induced from somatic cells such as fibroblasts bytransfection with one or more transcription factors. In some cases,cells are transformed with Oct3/4, Sox2, c-Myc and Klf4. The cells maybe additionally transfected with other genes, including transcriptionfactors and/or marker genes. The genes may be introduced using atransposon system such as the Cre/IoxP recombination system, or usingnon-integrating vectors in order to produce iPSCs free of exogenousreprogramming genes. Transfection may be achieved using viral vectors,such as a retrovirus. The virus may be an amphotropic virus. Once thecells have been transfected, they may be grown on feeder cells beforetransfer to an ESC culture medium.

iPS cells may be derived from any suitable cell type, including lung,foreskin fibroblasts, skin fibroblasts, keratinocytes, blood progenitorcells, bone marrow cells, hepatocytes, gastric epithelial cells,pancreatic cells, neural stem cells, B lymphocytes, ES derived somaticcells and embryonic fibroblasts. In some cases, the cells are not humandermal fibroblasts. The IPSCs may exhibit similar patterns of geneexpression and phenotype to ESCs.

Sources of Induced Pluripotent Stem Cells

Several methods have now been provided for the isolation of pluripotentstem cells that do not lead to the destruction of an embryo, e.g. bytransforming (inducing) adult somatic cells or germ cells. These methodsinclude:

1. Reprogramming by nuclear transfer. This technique involves thetransfer of a nucleus from a somatic cell into an oocyte or zygote. Insome situations this may lead to the creation of an animal-human hybridcell. For example, cells may be created by the fusion of a human somaticcell with an animal oocyte or zygote or fusion of a human oocyte orzygote with an animal somatic cell. 2. Reprogramming by fusion withembryonic stem cells. This technique involves the fusion of a somaticcell with an embryonic stem cell. This technique may also lead to thecreation of animal-human hybrid cells, as in 1 above. 3. Spontaneousre-programming by culture.

This technique involves the generation of pluripotent cells fromnon-pluripotent cells after long term culture. For example, pluripotentembryonic germ (EG) cells have been generated by long-term culture ofprimordial germ cells (PGC) (Matsui et al., Derivation of pluripotentialembryonic stem cells from murine primordial germ cells in culture. Cell70, 841-847, 1992, incorporated herein by reference). The development ofpluripotent stem cells after prolonged culture of bone marrow-derivedcells has also been reported (Jiang et al., Pluripotency of mesenchymalstem cells derived from adult marrow. Nature 418, 41-49, 2002,incorporated herein by reference). They designated these cellsmultipotent adult progenitor cells (MAPCs). Shinohara et al alsodemonstrated that pluripotent stem cells can be generated during thecourse of culture of germline stem (GS) cells from neonate mouse testes,which they designated multipotent germline stem (mGS) cells(Kanatsu-Shinohara et al.,

Generation of pluripotent stem cells from neonatal mouse testis. Cell119, 1001-1012, 2004). 4. Reprogramming by defined factors. For examplethe generation of iPS cells by the retrovirus-mediated introduction oftranscription factors (such as Oct-3/4, Sox2, c-Myc, and KLF4) intomouse embryonic or adult fibroblasts, e.g. as described above. Kaji etal (Virus-free induction of pluripotency and subsequent excision ofreprogramming factors. Nature. Online publication 1 Mar. 2009) alsodescribe the non-viral transfection of a single multiprotein expressionvector, which comprises the coding sequences of c-Myc, Klf4, Oct4 andSox2 linked with 2A peptides, that can reprogram both mouse and humanfibroblasts. iPS cells produced with this non-viral vector show robustexpression of pluripotency markers, indicating a reprogrammed stateconfirmed functionally by in vitro differentiation assays and formationof adult chimaeric mice. They succeeded in establishing reprogrammedhuman cell lines from embryonic fibroblasts with robust expression ofpluripotency markers.

Methods 1-4 are described and discussed by Shinya Yamanaka in Strategiesand New Developments in the Generation of Patient-Specific PluripotentStem Cells (Cell Stem Cell 1, July 2007 Elsevier Inc), incorporatedherein by reference. 5. Derivation of hESC lines from single blastomeresor biopsied blastomeres. See Klimanskaya I, Chung Y, Becker S, Lu S J,Lanza R. Human embryonic stem cell lines derived from singleblastomeres. Nature 2006; 444:512, Lei et al Xeno-free derivation andculture of human embryonic stem cells: current status, problems andchallenges. Cell Research (2007) 17:682-688, Chung Y, Klimanskaya I,Becker S, et al. Embryonic and extraembryonic stem cell lines derivedfrom single mouse blastomeres. Nature. 2006; 439:216-219. Klimanskaya I,Chung Y, Becker S, et al. Human embryonic stem cell lines derived fromsingle blastomeres. Nature. 2006; 444:481-485. Chung Y, Klimanskaya I,Becker S, et al. Human embryonic stem cell lines generated withoutembryo destruction. Cell Stem Cell. 2008; 2:113-117 and Dusko Ilic et al(Derivation of human tri embryonic stem cell lines from biopsiedblastomeres on human feeders with a minimal exposure to xenomaterials.Stem Cells And Development—paper in pre-publication), all incorporatedherein by reference. 6. hESC lines obtained from arrested embryos whichstopped cleavage and failed to develop to morula and blastocysts invitro. See Zhang X, Stojkovic P, Przyborski S, et al. Derivation ofhuman embryonic stem cells from developing and arrested embryos. StemCells 2006; 24:2669-2676 and Lei et al Xeno-free derivation and cultureof human embryonic stem cells: current status, problems and challenges.Cell Research (2007) 17:682-688, both incorporated herein by reference.7. Parthogenesis (or Parthenogenesis). This technique involves chemicalor electrical stimulation of an unfertilised egg so as to cause it todevelop into a blastomere from which embryonic stem cells may bederived. For example, see Lin et al. Multilineage potential ofhomozygous stem cells derived from metaphase II oocytes. Stem Cells.2003; 21(2): 152-61 who employed the chemical activation ofnonfertilized metaphase II oocytes to produce stem cells. 8. Stem cellsof fetal origin. These cells lie between embryonic and adult stem cellsin terms of potentiality and may be used to derive pluripotent ormultipotent cells. Human umbilical-cord-derived fetal mesenchymal stemcells (UC fMSCs) expressing markers of pluripotency (including Nanog,Oct-4, Sox-2, Rex-1, SSEA-3, SSEA-4, Tra-1-60, and Tra-1-81, minimalevidence of senescence as shown by .beta.-galactosidase staining, andthe consistent expression of telomerase activity) have been successfullyderived by Chris H. Jo et al (Fetal mesenchymal stem cells derived fromhuman umbilical cord sustain primitive characteristics during extensiveexpansion. Cell Tissue Res (2008) 334:423-433, incorporated herein byreference). Winston Costa Pereira et al (Reproducible methodology forthe isolation of mesenchymal stem cells from human umbilical cord andits potential for cardiomyocyte generation J Tissue Eng Regen Med 2008;2: 394-399, incorporated herein by reference) isolated a pure populationof mesenchymal stem cells from Wharton's jelly of the human umbilicalcord. Mesenchymal stem cells derived from Wharton's jelly are alsoreviewed in Troyer & Weiss (Concise Review: Wharton's Jelly-DerivedCells Are a primitive Stromal Cell Population. Stem Cells2008:26:591-599) Kim et al (Ex vivo characteristics of human amnioticmembrane-derived stem cells. Cloning Stem Cells 2007 Winter;9(4):581-94, incorporated herein by reference) succeeded in isolatinghuman amniotic membrane-derived mesenchymal cells from human amnioticmembranes. Cimbilical cord is a tissue that is normally discarded andstem cells derived from this tissue have tended not to attract moral orethical objection.

Induced pluripotent stem cells have the advantage that they can beobtained by a method that does not cause the destruction of an embryo,more particularly by a method that does not cause the destruction of ahuman or mammalian embryo. As such, aspects of the invention may beperformed or put into practice by using cells that have not beenprepared exclusively by a method which necessarily involves thedestruction of human or animal embryos from which those cells may bederived. This optional limitation is specifically intended to takeaccount of Decision G0002/06 of 25 Nov. 2008 of the Enlarged Board ofAppeal of the European Patent Office.

Mesenchymal Stem Cells

Mesenchymal stem cells are known as being multipotent and exhibit thepotential for differentiation into different cells/tissue lineages,including cartilage, bone, adipose tissue, tendon, and ligament. Thesemultipotent mesenchymal progenitor cells are denoted as stromal ormesenchymal stem cells. Bone marrow contains two main cell types:hematopoietic cells and stromal cells. The stem cells for nonhematopoietic tissues are referred as mesenchymal cells because of theirability to differentiate as mesenchymal or stromal cells.

Accordingly, in this specification mesenchymal stem cells (MSCs) refersto multipotent stem cells capable of differentiation into osteoblasts,chondrocytes, myocytes, adipocytes and endothelium. In thisspecification MSCs particularly refers to multipotent stem cells capableof differentiation into osteoblasts as part of the process of formationof bone.

Mesenchymal cells are easily obtainable from bone marrow by minimallyinvasive techniques and can be expanded in culture and permitted todifferentiate into the desired lineage. Differentiation can be inducedby the application of specific growth factors. The transforming growthfactor beta (TGF-beta) superfamily member proteins such as the bonemorphogenetic proteins (BMPs) are important factors of chondrogenic andosteogenic differentiation of mesenchymal stem cells. Suitable MSCs maybe obtained or derived from bone marrow mononuclear cells (BMMNCs)collected from aspirates of bone marrow (e.g. Wexler et al. Adult bonemarrow is a rich source of human mesenchymal ‘stem’ cells but umbilicalcord and mobilized adult blood are not. HAEMOPOIESIS AND LEUCOCYTESBritish Journal of Haematology 121(2):368-374, April 2003.) or Wharton'sJelly of the umbilical cord (e.g. Ta et al. Long-term Expansion andPluripotent Marker Array Analysis of Wharton's Jelly-Derived MesenchymalStem Cells. Stem Cells Dev. 2009 Jul. 20 (Epub)).

Differentiation of MSCs to the osteogenic lineage may be achieved byculture in osteogenic medium. For example, MSCs are seeded at 3,000/cm.sup.2 in maintenance medium (DMEM, 1 g/l glucose, 10% FCS, 2 mML-glutamine, 50 U/ml penicillin and 50 U/ml streptomycin) in 6-well,12-well and chamber slides for 24 h before changing to osteogenic media(maintenance medium, 10 nM dexamethasone, 25.mu.g/ml ascorbic acid and10 mM beta-glycerophosphate). Cells are then maintained for up to 28days with a media change every 3-4 days. After 14 days cells in thechamber slides may be fixed in 4% PFA and stored at 4.degree. C. in PBSfor immunohistochemistry. After 14 and 28 days the cells are stainedwith alizarin red S for calcium, and von Kossa for calcium phosphate.RNA may also be extracted for analysis using the Nucleospin RNAextraction kit according to the manufacturer's instructions (MachereyNagel) and protein samples may be extracted for analysis.

Differentiation of MSCs to the adipogenic lineage may be achieved byculture in adipogenic medium. For example, MSCs are seeded at18,000/cm.sup.2 in maintenance medium and incubated as above for 2 days.

Media is removed and cells are washed once in PBS before the addition ofadipogenic maintenance media (DMEM, 4.5 g/l glucose, 10% FCS,L-glutamine and penicillin and streptomycin) or adipogenic media(adipogenic maintenance media with 10.mu.g/ml insulin, 115 microg/mlmethyl-isobutylxanthine, 1.mu.M dexamethasone and 20.mu.Mindomethazine). Cells are then maintained for up to 28 days with a mediachange every 3-4 days. After 14 and 28 days the cells may be stainedwith oil-red-0 to stain the lipid droplets. RNA and protein may also beextracted for analysis.

Differentiation of MSCs to the chondrogenic lineage may be achieved byculture in chrondrogenic medium. For example, MSCs are counted andresuspended at 5.times. 10.sup.5 cells/ml in chondrogenic media (DMEMwith Cambrex chondrogenic single aliquots) with or without 10 ng/mlTGF.quadrature.3 (Cambrex) and then 500 ml aliquots were put into 15 mltubes before centrifugation at 150.times.g at room temperature for 10min and incubated at 37° C. for 2 days. After two days the tubes willcontain loose round pellets. Pellets are maintained for 21 days with amedia change every 3-4 days before RNA is isolated using Trizol(Invitrogen) or cell pellets are fixed in 4% PFA and embedded forcryosectioning Serial sections are made before slides are stored at −80°C. for immunohistochemistry.

When osteogenic and adipogenic differentiation are investigated underconfluent conditions, cells may be seeded at 30,000/cm² and allowed toreach confluence before switching to the relevant differentiation mediaand cultured as above.

Culture of Stem Cells

Any suitable method of culturing stem cells may be used, and anysuitable container may be used to propagate stem cells. Suitablecontainers include those described in US Patent PublicationUS2007/0264713 (Terstegge).

Containers may include bioreactors and spinners, for example. A“bioreactor” is a container suitable for the cultivation of eukaryoticcells, for example animal cells or mammalian cells, such as in a largescale. A typical cultivation volume of a regulated bioreactor is between20 ml and 500 ml. The bioreactor may comprise a regulated bioreactor, inwhich one or more conditions may be controlled or monitored, forexample, oxygen partial pressure. Devices for measuring and regulatingthese conditions are known in the art. For example, oxygen electrodesmay be used for oxygen partial pressure. The oxygen partial pressure canbe regulated via the amount and the composition of the selected gasmixture (e.g., air or a mixture of air and/or oxygen and/or nitrogenand/or carbon dioxide). Suitable devices for measuring and regulatingthe oxygen partial pressure are described by Bailey, J E. (Bailey, J E.,Biochemical Engineering Fundamentals, second edition, McGraw-Hill, Inc.ISBN 0-07-003212-2 Higher Education, (1986)) or Jackson A T. Jackson AT., Verfahrenstechnik in der Biotechnologie, Springer, ISBN 3540561900(1993)).

Other suitable containers include spinners. Spinners are regulated orunregulated bioreactors, which can be agitated using various agitatormechanisms, such as glass ball agitators, impeller agitators, and othersuitable agitators. The cultivation volume of a spinner is typicallybetween 20 ml and 500 ml. Roller bottles are round cell culture flasksmade of plastic or glass having a culture area of between 400 and 2000cm. sup.2. The cells are cultivated along the entire inner surface ofthese flasks; the cells are coated with culture medium accomplished by a“rolling” motion, i.e. rotating the bottles about their own individualaxis.

Alternatively, culture may be static, i.e. where active agitation of theculture/culture media is not employed. By reducing agitation of theculture, aggregates of cells may be allowed to form. Whilst someagitation may be employed to encourage distribution and flow of theculture media over the cultured cells this may be applied so as not tosubstantially disrupt aggregate formation. For example, a low rpmagitation, e.g. less than 30 rpm or less than 20 rpm, may be employed.

Propagation with Passage. Methods of cell culture may comprisepassaging, or splitting during culture. The methods may involvecontinuous or continual passage. Cells in culture may be dissociatedfrom the substrate or flask, and “split”, subcultured or passaged, bydilution into tissue culture medium and replating/re-culturing. The term“passage” may generally refer to the process of taking an aliquot of acell culture, dissociating the cells completely or partially, dilutingand inoculating into medium. The passaging may be repeated one or moretimes. The aliquot may comprise the whole or a portion of the cellculture. The cells of the aliquot may be completely, partially or notconfluent. The passaging may comprise at least some of the followingsequence of steps: aspiration, rinsing, trypsinization, incubation,dislodging, quenching, re-seeding and aliquoting. The protocol publishedby the Hedrick Lab, UC San Diego may be used.

The cells may be dissociated by any suitable means, such as mechanicalor enzymatic means known in the art. The cells may be broken up bymechanical dissociation, for example using a cell scraper or pipette.The cells may be dissociated by sieving through a suitable sieve size,such as through 100 micron or 500 micron sieves. The cells may be splitby enzymatic dissociation, for example by treatment with collagenase ortrypLE harvested. The dissociation may be complete or partial. Thedilution may be of any suitable dilution. The cells in the cell culturemay be split at any suitable ratio. For example, the cells may be splitat a ratio of 1:2 or more, 1:3 or more, 1:4 or more or 1:5 or more.Thus, stem cells may be passaged for 1 passage or more. For example,stem cells may be passaged for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21,22, 23,24, 25 passages or more. Passagesmay be expressed as generations of cell growth. Stem cells may bepropagated for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25 generations or more. Stem cells maybe propagated for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25 cell doublings or more Co-Culture andFeeders Methods may comprise culturing stem cells in the presence orabsence of co-culture. The term “co-culture” refers to a mixture of twoor more different kinds of cells that are grown together. The two ormore different kinds of cells may be grown on the same surfaces, such asparticles or cell container surfaces, or on different surfaces. Thedifferent kinds of cells may be grown on different particles. Feedercells may mean cells which are used for or required for cultivation ofcells of a different type. In the context of stem cell culture, feedercells have the function of securing the survival, proliferation, andmaintenance of cell pluripotency or multipotency. Cellpluripotency/multipotency may be ensured by directly co-cultivating thefeeder cells. For example, the inner surface of the container such as aculture dish may be coated with a feeder cell layer. The feeder cellsrelease nutrients into the culture medium. Alternatively, or inaddition, the feeder cells may be cultured in a medium to condition it.The conditioned medium may be used to culture the stem cells. Thus,arrangements in which feeder cells are absent or not required are alsopossible.

The invention provides the treatment of stem cells with GR antagonist toyield GR antagonist treated stem cells.

Osteoporosis and Bone Related Injuries

Glucocorticoids (GCs) are central to the treatment of inflammatory andimmune disorders. These steroids, however, profoundly impact theskeleton, particularly when administered for prolonged periods. In fact,high-dose GC therapy is almost universally associated with bone loss,causing one of the most crippling forms of osteoporosis. Despite thefrequency and severity of GC-induced osteoporosis, its treatment is lessthan satisfactory, suggesting that its pathogenesis is incompletelyunderstood. The present invention provides a method and treatment forthese disorders as well as the rapid site-specific bone growth by acombination cortisol blockers i.e. The present invention relates to theuse of a GCR antagonist, such as for example, ORG34517, PT150, PT155,PT156, PT157, PT158, TCY1, combinations thereof, and pharmaceuticallyacceptable salts thereof, for the prevention or treatment of and theoptional use of bone marrow irrigation, bone compatible cement and PTHtherapy.

The present invention relates to the use of GR antagonists or GRantagonist treated stem cells for the prevention or treatment of stressinduced osteoporosis and the rapid healing of bone related injuries

The present invention is concerned with the therapeutic use (human andveterinary) of GR antagonists or GR antagonist treated stem cells totreat bone fracture. GR antagonists and GR antagonist treated stem cellsare reported here to augment wound healing in bone. GR antagonistsstimulate bone regeneration following injury and contribute to improvedwound healing in bone. GR antagonists and GR antagonist treated stemcells provide improvements in the speed of bone fracture repair enablinga reduction in the recovery time from injury.

Bone fracture is a medical condition. In this application “fracture”includes damage or injury to bone in which a bone is cracked, broken orchipped. A break refers to discontinuity in the bone. A fracture may becaused by physical impact, or mechanical stress or by medical conditionssuch as osteoporosis or osteoarthritis.

Orthopedic classification of fractures includes closed or open andsimple or multi-fragmentary fractures. In closed fractures the skinremains intact, whilst in an open fracture the bone may be exposedthrough the wound site, which brings a higher risk of infection. Simplefractures occur along a single line, tending to divide the bone in two.Multi-fragmentary fractures spilt the bone into multiple pieces.

Other fracture types include, compression fracture, compacted fracture,spiral fracture, complete and incomplete fractures, transverse, linearand oblique fractures and comminuted fractures. In most subjects bonehealing (fracture union) occurs naturally and is initiated followinginjury. Bleeding normally leads to clotting and attraction of whiteblood cells and fibroblasts, followed by production of collagen fibres.This is followed by bone matrix (calcium hydroxyapatite) deposition(mineralisation) transforming the collagen matrix into bone. Immaturere-generated bone is typically weaker than mature bone and over time theimmature bone undergoes a process of remodelling to produce mature“lamellar” bone. The complete bone healing process takes considerabletime, typically many months.

Bones in which fractures occur and which may benefit from treatmentusing GR antagonists include all bone types, particularly all mammalianbones including, but not limited to, long bones (e.g. femur, humerus,phalanges), short bones (e.g. carpals, tarsals), flat bones (e.g.cranium, ribs, scapula, sternum, pelvic girdle), irregular bones (e.g.vertebrae), sesamoid bones (e.g. patella). Bone fracture also includespathological porosity, such as that exhibited by subjects withosteoporosis. GR antagonists and GR antagonist treated stem cells andpharmaceutical compositions and medicaments comprising GR antagonistsand GR antagonist treated stem cells are provided for use in a method oftreatment of bone fracture in a mammalian subject.

Treatment may comprise wound healing in bone. The treatment may involverepair, regeneration and growth of bone. GR antagonists and GRantagonist treated stem cells facilitate fracture repair by facilitatingnew bone growth. GR antagonists act to improve the speed of fracturerepair enabling bone healing to occur faster leading to improvedrecovery time from injury.

Treatment may lead to improved bone strength. Treatment may also includetreatment of osteoporosis or osteoarthritis. Administration of GRantagonists and GR antagonist treated stem cells may for example be tothe tissue surrounding the fracture. This may include administrationdirectly to bone tissue in which the fracture has occurred.Administration may be to connective tissue surrounding the bone orfracture or to vasculature (e.g. blood vessels) near to and supplyingthe bone. Administration may be directly to the site of injury and maybe to a callus formed by initial healing of the wound.

Medicaments and pharmaceutical compositions according to the presentinvention may be formulated for administration by a number of routes. GRantagonists and GR antagonist treated stem cells may be formulated influid or liquid form for injection, or as part of a gel suitable forapplication to bone or other tissue surrounding the fracture.

Administration is preferably in a “therapeutically effective amount”,this being sufficient to improve healing of the bone fracture comparedto a corresponding untreated fracture or to a fracture treated with GRantagonist treated stem cells obtained from culture in controlconditions. The actual amount administered, and rate and time-course ofadministration, will depend on the nature and severity of the fracture.Prescription of treatment, e.g. decisions on dosage etc, is within theresponsibility of general practitioners and other medical doctors, andwill typically take account of the nature of the fracture, the conditionof the individual patient, the site of delivery, the method ofadministration and other factors known to practitioners. Single ormultiple administrations of GR antagonists or GR antagonist treated stemcell doses may be administered in accordance with the guidance of theprescribing medical practitioner. Examples of the techniques andprotocols mentioned above can be found in Remington's PharmaceuticalSciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

GR antagonists or GR antagonist treated stem cells may be used to treatbone fracture alongside other treatments, such as administration of painrelieving or anti-inflammatory medicaments, immobilisation and settingof the bone, e.g. immobilising the injured limb in a plaster cast,surgical intervention, e.g. to re-set a bone or move a bone to correctdisplacement, angulation or dislocation. If surgery is required GRantagonists or GR antagonist treated stem cells may be administereddirectly to (e.g. applied to) the fracture during the surgicalprocedure.

Pharmaceutical compositions and medicaments of the invention may takethe form of a biomaterial that is coated and/or impregnated with GRantagonists or GR antagonist treated stem cells. An implant orprosthesis may be formed from the biomaterial. Such implants orprostheses may be surgically implanted to assist in bone growth,regeneration, restructuring and/or remodelling.

GR antagonists or GR antagonist treated stem cells may be applied toimplants or prostheses to accelerate new bone formation at a desiredlocation. The biomaterial may be coated or impregnated with GRantagonists or GR antagonist treated stem cells. Impregnation maycomprise contacting the GR antagonists with the biomaterial such thatthey are allowed to be adsorbed and/or absorbed onto and/or into thebiomaterial. Coating may comprise adsorbing the GR antagonists or GRantagonist treated stem cells onto the surface of the biomaterial.Coating or impregnation of the biomaterial may involve seeding GRantagonists or GR antagonist treated stem cells onto or into thebiomaterial.

The biomaterial should allow the coated or impregnated GR antagonists orGR antagonist treated stem cells to be released from the biomaterialwhen administered to or implanted in the subject. Biomaterial releasekinetics may be altered by altering the structure, e.g. porosity, of thebiomaterial.

In addition to coating or impregnating a biomaterial with GR antagonistsor GR antagonist treated stem cells, one or more biologically activemolecules may be impregnated or coated on the biomaterial. For example,at least one chosen from the group consisting of: BMP-2, BMP-4, OP-1,FGF-1, FGF-2, TGF-beta1, TGF-beta2, TGF-beta3; VEGF; collagen; laminin:fibronectin; vitronectin. In addition or alternatively to the abovebioactive molecules, one or more bisphosphonates may be impregnated orcoated onto the biomaterial along with GR antagonists. Examples ofuseful bisphosphonates may include at least one chosen from the groupconsisting of: etidronate; clodronate; alendronate; pamidronate;risedronate; zoledronate. Optionally, GR antagonists or GR antagonisttreated stem cells is excluded from being impregnated or coated on thebiomaterial.

Biomaterials coated or impregnated with GR antagonists or GR antagonisttreated stem cells may be useful in both medical and veterinarypurposes. It will be appreciated that the present invention may improvethe quality of life of a patient or potentially extend the life of ananimal, for example a valuable race horse for use in breeding.

The biomaterial provides a scaffold or matrix support. The biomaterialmay be suitable for implantation in tissue, or may be suitable foradministration (e.g. as microcapsules in solution). The implant orprosthesis should be biocompatible, e.g. non-toxic and of lowimmunogenicity (most preferably non-immunogenic). The biomaterial may bebiodegradable such that the biomaterial degrades as wound healingoccurs, ultimately leaving only the regenerated bone in situ in thesubject. Alternatively a non-biodegradable biomaterial may be used, e.g.to guide bone regeneration over a large discontinuity and/or to act as astructural support during bone healing, with surgical removal of thebiomaterial being an optional requirement after successful woundhealing.

Biomaterials may be soft and/or flexible, e.g. hydrogels, fibrin web ormesh, or collagen sponges. A “hydrogel” is a substance formed when anorganic polymer, which can be natural or synthetic, is set or solidifiedto create a three-dimensional open-lattice structure that entrapsmolecules of water or other solutions to form a gel. Solidification canoccur by aggregation, coagulation, hydrophobic interactions orcross-linking.

Alternatively biomaterials may be relatively rigid structures, e.g.formed from solid materials such as plastics or biologically inertmetals such as titanium. The biomaterial may have a porous matrixstructure which may be provided by a crosslinked polymer. The matrix ispreferably permeable to nutrients and growth factors required for bonegrowth.

Matrix structures may be formed by crosslinking fibres, e.g. fibrin orcollagen, or of liquid films of sodium alginate, chitosan, or otherpolysaccharides with suitable crosslinkers, e.g. calcium salts,polyacrylic acid, heparin. Alternatively scaffolds may be formed as agel, fabricated by collagen or alginates, crosslinked using wellestablished methods known to those skilled in the art.

Suitable polymer materials for matrix formation include, but are notlimited by, biodegradable/bioresorbable polymers which may be chosenfrom the group of agarose, collagen, fibrin, chitosan, polycaprolactone,poly(DL-lactide-co-caprolactone),poly(L-lactide-co-caprolactone-co-glycolide), polyglycolide,polylactide, polyhydroxyalcanoates, co-polymers thereof, ornon-biodegradable polymers which may be chosen from the group of:cellulose acetate; cellulose butyrate, alginate, polysulfone,polyurethane, polyacrylonitrile, sulfonated polysulfone, polyamide,polyacrylonitrile, polymethylmethacrylate, co-polymers thereof.

Collagen is a promising material for matrix construction owing to itsbiocompatibility and favourable property of supporting cell attachmentand function (U.S. Pat. No. 5,019,087; Tanaka, S.; Takigawa, T.;Ichihara, S. & Nakamura, T. Mechanical properties of the bioabsorbablepolyglycolic acid-collagen nerve guide tube Polymer Engineering &Science 2006, 46, 1461-1467). Clinically acceptable collagen sponges areone example of a matrix and are well known in the art (e.g. from IntegraLife Sciences).

Fibrin scaffolds (e.g. fibrin glue) provide an alternative matrixmaterial. Fibrin glue enjoys widespread clinical application as a woundsealant, a reservoir to deliver growth factors and as an aid in theplacement and securing of biological implants (Rajesh Vasita, DhirendraS Katti. Growth factor delivery systems for tissue engineering: amaterials perspective. Expert Reviews in Medical Devices. 2006; 3(1):29-47; Wong C, Inman E, Spaethe R, Helgerson S. Thromb. Haemost. 200389(3): 573-582; Pandit A S, Wilson D J, Feldman D S. Fibrin scaffold asan effective vehicle for the delivery of acidic growth factor (FGF-1).J. Biomaterials Applications. 2000; 14(3); 229-242; DeBlois Cote M F.Doillon C J. Heparin-fibroblast growth factor fibrin complex: in vitroand in vivo applications to collagen based materials. Biomaterials.1994; 15(9): 665-672.).

Luong-Van et al (In vitro biocompatibility and bioactivity ofmicroencapsulated heparan sulphate Biomaterials 28 (2007) 2127-2136),incorporated herein by reference, describes prolonged localised deliveryof HS from polycaprolactone microcapsules. A further example of abiomaterial is a polymer that incorporates hydroxyapatite or hyaluronicacid.

The biomaterial can be supplemented with additional cells. For example,one can “seed” the biomaterial (or cosynthesise it) with fibroblastderived feeder cells, which may be useful for supporting growth andmaintenance of the GR antagonists.

The subject to be treated may be any animal or human. The subject ispreferably mammalian. In some embodiments the subject is a human. Inother embodiments the subject is an animal, more preferably a nonhumanmammal. The non-human mammal may be a domestic pet, or animal kept forcommercial purposes, e.g. a race horse, or farming livestock such aspigs, sheep or cattle. As such the invention may have veterinaryapplications. Non-human mammals include rabbits, guinea pigs, rats, miceor other rodents (including any animal in the order Rodentia), cats,dogs, pigs, sheep, goats, cattle (including cows or any animal in theorder Bos), horse (including any animal in the order Equidae), donkey,and non-human primates. The subject may be male or female. The subjectmay be a patient.

Wound Healing and Transplants

While elevated cortisol plays important roles in physiologic homeostasisin the face of extreme physical and emotional stress, it can havenegative effects on wound healing, by inhibiting cells important towound repair (including stem cells) through binding to theirglucocorticoid receptors (GR).

Application of a GR antagonist or active agent such as ORG 34517, PT150,PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof can improve wound healing insuch circumstances. However, blockade of systemic cortisol binding to GRmust be avoided. Thus, the present inventions provides for the localapplication of PT155, PT156, or PT157 to wounds (by topical applicationin cutaneous wounds or by direct injection or local vascular infusion)will help in wound repair in the face of acute physical trauma (e.g. warwounds) while at the same time preserving the systemic homeostasis towhich cortisol contributes. Application of the same to stem celltherapies is also provided by pre-treatment of transplantable stem cellswith ORG 34517, PT150, PT155, PT156, PT157, PT158, TCY1, combinationsthereof, and pharmaceutically acceptable salts thereof. In addition, theinvention provides for pre-treatment of an implantable organ prior toimplantation in the recipient. The invention provides for treatment ofan implantable organ during implantation in the recipient.

Perfusion Systems

A perfusion system for cells may be used to expose a tissue or organ toa GR antagonist in the form of a liquid or a semi-solid. Perfusionrefers to continuous flow of a solution through or over a population ofcells. It implies the retention of the cells within the culture unit asopposed to continuous-flow culture, which washes the cells out with thewithdrawn media (e.g., chemostat). Perfusion allows for better controlof the culture environment (pH, pO.sub.2, nutrient levels, GR antagonistlevels, etc.) and is a means of significantly increasing the utilizationof the surface area within a culture for cell attachment.

The technique of perfusion was developed to mimic the cells milieu invivo where cells are continuously supplied with blood, lymph, or otherbody fluids. Without perfusion of a physiological nutrient solution,cells in culture go through alternating phases of being fed and starved,thus limiting full expression of their growth and metabolic potential.In the context of the present invention, a perfusion system may also beused to perfuse cells with an GR antagonist to induce stasis.

Those of skill in the art are familiar with perfusion systems, and thereare a number of perfusion systems available commercially. Any of theseperfusion systems may be employed in the present invention. One exampleof a perfusion system is a perfused packed-bed reactor using a bedmatrix of a non-woven fabric (CelliGen®, New Brunswick Scientific,Edison, N.J.; Wang et al., 1992; Wang et al., 1993; Wang et al., 1994).Briefly described, this reactor comprises an improved reactor forculturing of both anchorage- and nonanchorage-dependent cells. Thereactor is designed as a packed bed with a means to provide internalrecirculation. Preferably, a fiber matrix carrier is placed in a basketwithin the reactor vessel. A top and bottom portion of the basket hasholes, allowing the medium to flow through the basket. A speciallydesigned impeller provides recirculation of the medium through the spaceoccupied by the fiber matrix for assuring a uniform supply of nutrientand the removal of wastes. This simultaneously assures that a negligibleamount of the total cell mass is suspended in the medium. Thecombination of the basket and the recirculation also provides abubble-free flow of oxygenated medium through the fiber matrix. Thefiber matrix is a non-woven fabric having a “pore” diameter of from 10micrometer to 100 micrometer, providing for a high internal volume withpore volumes corresponding to 1 to 20 times the volumes of individualcells.

The perfused packed-bed reactor offers several advantages. With a fibermatrix carrier, the cells are protected against mechanical stress fromagitation and foaming. The free medium flow through the basket providesthe cells with optimum regulated levels of oxygen, pH, and nutrients.Products can be continuously removed from the culture and the harvestedproducts are free of cells and can be produced in low-protein medium,which facilitates subsequent purification steps. This technology isexplained in detail in WO 94/17178 (Aug. 4, 1994, Freedman et al.),which is hereby incorporated by reference in its entirety.

The Cellcube® (Corning-Costar) module provides a large styrenic surfacearea for the immobilization and growth of substrate attached cells. Itis an integrally encapsulated sterile single-use device that has aseries of parallel culture plates joined to create thin sealed laminarflow spaces between adjacent plates.

The Cellcube® module has inlet and outlet ports that are diagonallyopposite each other and help regulate the flow of media. During thefirst few days of growth the culture is generally satisfied by the mediacontained within the system after initial seeding. The amount of timebetween the initial seeding and the start of the media perfusion isdependent on the density of cells in the seeding inoculum and the cellgrowth rate. The measurement of nutrient concentration in thecirculating media is a good indicator of the status of the culture. Whenestablishing a procedure it may be necessary to monitor the nutrientscomposition at a variety of different perfusion rates to determine themost economical and productive operating parameters.

Other commercially available perfusion systems include, for example,CellPerf® (Laboratories MABIO International, Tourcoing, France) and theStovall Flow Cell (Stovall Life Science, Inc., Greensboro, N.C.). Thetiming and parameters of the production phase of cultures depends on thetype and use of a particular cell line. Many cultures require adifferent media for production than is required for the growth phase ofthe culture. The transition from one phase to the other will likelyrequire multiple washing steps in traditional cultures. However, one ofthe benefits of a perfusion system is the ability to provide a gentletransition between various operating phases. The perfusion system canalso facilitate the transition from a growth phase to a static phaseinduced by an GR antagonist. Likewise, the perfusion system canfacilitate the transition from a static phase to a growth phase byreplacing the solution comprising an GR antagonist with, for example, aphysiological nutrient media.

Formulations

The compounds of the invention may be administered enterally orparenterally. Mixed with pharmaceutically suitable auxiliaries, e.g., asdescribed in the standard reference, Gennaro et al., Remington'sPharmaceutical Sciences. The compounds may be compressed into soliddosage units, such as pills, tablets, or be processed into capsules orsuppositories. By means of pharmaceutically suitable liquids thecompounds can also be applied in the form of a solution, suspension,emulsion, e.g. for use as an injection preparation or eye drops, or as aspray, e.g. for use as a nasal spray.

For making dosage units, e.g., tablets, the use of conventionaladditives such as fillers, colorants, polymeric binders and the like iscontemplated. In general, any pharmaceutically acceptable additive whichdoes not interfere with the function of the active compounds can beused. Suitable carriers with which the compositions can be administeredinclude lactose, starch, cellulose derivatives and the like, or mixturesthereof, used in suitable amounts.

Dosage Forms

The compositions of the present invention can be processed byagglomeration, air suspension chilling, air suspension drying, balling,coacervation, coating, comminution, compression, cryopelletization,encapsulation, extrusion, wet granulation, dry granulation,homogenization, inclusion complexation, lyophilization, melting,microencapsulation, mixing, molding, pan coating, solvent dehydration,sonication, spheronization, spray chilling, spray congealing, spraydrying, or other processes known in the art. The compositions can beprovided in the form of a minicapsule, a capsule, a smart capsule, atablet, an implant, a troche, a lozenge (minitablet), a temporary orpermanent suspension, an ovule, a suppository, a wafer, a chewabletablet, a quick or fast dissolving tablet, an effervescent tablet, abuccal or sublingual solid, a granule, a film, a sprinkle, a pellet, abead, a pill, a powder, a triturate, a platelet, a strip or a sachet.Compositions can also be administered as a “dry syrup”, where thefinished dosage form is placed directly on the tongue and swallowed orfollowed with a drink or beverage.

These forms are well known in the art and are packaged appropriately.The compositions can be formulated for oral, nasal, buccal, ocular,urethral, transmucosal, vaginal, topical or rectal delivery.

The pharmaceutical composition can be coated with one or more entericcoatings, seal coatings, film coatings, barrier coatings, compresscoatings, fast disintegrating coatings, or enzyme degradable coatings.Multiple coatings can be applied for desired performance. Further, thedosage form can be designed for immediate release, pulsatile release,controlled release, extended release, delayed release, targeted release,synchronized release, or targeted delayed release. Forrelease/absorption control, solid carriers can be made of variouscomponent types and levels or thicknesses of coats, with or without anactive ingredient. Such diverse solid carriers can be blended in adosage form to achieve a desired performance. The definitions of theseterms are known to those skilled in the art. In addition, the dosageform release profile can be affected by a polymeric matrix composition,a coated matrix composition, a multiparticulate composition, a coatedmultiparticulate composition, an ion-exchange resin-based composition,an osmosis-based composition, or a biodegradable polymeric composition.Without wishing to be bound by theory, it is believed that the releasemay be effected through favorable diffusion, dissolution, erosion,ion-exchange, osmosis or combinations thereof.

When formulated as a capsule, the capsule can be a hard or soft gelatincapsule, a starch capsule, or a cellulosic capsule. Although not limitedto capsules, such dosage forms can further be coated with, for example,a seal coating, an enteric coating, an extended release coating, or atargeted delayed release coating. These various coatings are known inthe art, but for clarity, the following brief descriptions are provided:seal coating, or coating with isolation layers: Thin layers of up to 20microns in thickness can be applied for variety of reasons, includingfor particle porosity reduction, to reduce dust, for chemicalprotection, to mask taste, to reduce odor, to minimize gastrointestinalirritation, etc. The isolating effect is proportional to the thicknessof the coating. Water soluble cellulose ethers are preferred for thisapplication. HPMC and ethyl cellulose in combination, or Eudragit E100,may be particularly suitable for taste masking applications. Traditionalenteric coating materials listed elsewhere can also be applied to forman isolating layer.

Extended release coatings are designed to effect delivery over anextended period of time. The extended release coating is apH-independent coating formed of, for example, ethyl cellulose,hydroxypropyl cellulose, methylcellulose, hydroxymethyl cellulose,hydroxyethyl cellulose, acrylic esters, or sodium carboxymethylcellulose. Various extended release dosage forms can be readily designedby one skilled in art to achieve delivery to both the small and largeintestines, to only the small intestine, or to only the large intestine,depending upon the choice of coating materials and/or coating thickness.

Enteric coatings are mixtures of pharmaceutically acceptable excipientswhich are applied to, combined with, mixed with or otherwise added tothe carrier or composition. The coating may be applied to a compressedor molded or extruded tablet, a gelatin capsule, and/or pellets, beads,granules or particles of the carrier or composition. The coating may beapplied through an aqueous dispersion or after dissolving in appropriatesolvent. Additional additives and their levels, and selection of aprimary coating material or materials will depend on the followingproperties: 1. resistance to dissolution and disintegration in thestomach; 2. impermeability to gastric fluids and drug/carrier/enzymewhile in the stomach; 3. ability to dissolve or disintegrate rapidly atthe target intestine site; 4. physical and chemical stability duringstorage; 5. non-toxicity; 6. easy application as a coating (substratefriendly); and 7. economical practicality.

Dosage forms of the compositions of the present invention can also beformulated as enteric coated delayed release oral dosage forms, i.e., asan oral dosage form of a pharmaceutical composition as described hereinwhich utilizes an enteric coating to affect release in the lowergastrointestinal tract. The enteric coated dosage form may be acompressed or molded or extruded tablet/mold (coated or uncoated)containing granules, pellets, beads or particles of the activeingredient and/or other composition components, which are themselvescoated or uncoated. The enteric coated oral dosage form may also be acapsule (coated or uncoated) containing pellets, beads or granules ofthe solid carrier or the composition, which are themselves coated oruncoated.

Delayed release generally refers to the delivery so that the release canbe accomplished at some generally predictable location in the lowerintestinal tract more distal to that which would have been accomplishedif there had been no delayed release alterations. The preferred methodfor delay of release is coating. Any coatings should be applied to asufficient thickness such that the entire coating does not dissolve inthe gastrointestinal fluids at pH below about 5, but does dissolve at pHabout 5 and above. It is expected that any anionic polymer exhibiting apH-dependent solubility profile can be used as an enteric coating in thepractice of the present invention to achieve delivery to the lowergastrointestinal tract. Polymers for use in the present invention areanionic carboxylic polymers.

Shellac, also called purified lac, a refined product obtained from the,resinous secretion of an insect. This coating dissolves in media ofpH>7.Colorants, detackifiers, surfactants, antifoaming agents, lubricants,stabilizers such as hydroxy propyl cellulose, acid/base may be added tothe coatings besides plasticizers to solubilize or disperse the coatingmaterial, and to improve coating performance and the coated product.In carrying out the method of the present invention, the combination ofthe invention may be administered to mammalian species, such as dogs,cats, humans, etc. and as such may be incorporated in a conventionalsystemic dosage form, such as a tablet, capsule, elixir or injectable.The above dosage forms will also include the necessary carrier material,excipient, lubricant, buffer, antibacterial, bulking agent (such asmannitol), antioxidants (ascorbic acid of sodium bisulfite) or the like.

The invention provides a smart pill or smart capsule, which is, in anexemplary embodiment, an ingestible drug delivery device configured forwireless communication with other ingestible drug delivery devices, saiddrug delivery device comprising: a capsule body comprising: a sensor forsensing at least one biologic condition within a patient and providing afirst signal representative thereof; a bioactive substance modulecomprising a container for holding a volume and/or quantity of bioactivesubstance therein and a microactuator for dispensing said bioactivesubstance from said container to a location outside of said capsulebody; an electronics module, coupled to said sensor and said bioactivesubstance module, said electronics module comprising a processor, atransponder and a memory (e.g., flash, OTP, etc.), said memorycomprising data selected from the group consisting of: (a) data relatedto the patient who is permitted to ingest said ingestible drug medicaldevice; (b) data related to said bioactive substance; (c) data relatedto a healthcare provider that enabled said electronics module; (d) datarelated to said sensor; (e) data related to the provenance of saidingested drug medical device; (f) combinations thereof, a power sourcecoupled to said sensor, said bioactive substance module and saidelectronics module; and wherein said processor controls said transponderto transmit at least one wireless signal and to receive at least onewireless signal from at least one other ingestible medical device, andwherein said processor receives said first signal and analyzes saidfirst signal with all of said data along with said received at least onewireless signal for controlling said microactuator for dispensing saidbioactive substance.

In exemplary embodiments, ORG 34517, PT150, PT155, PT156, PT157, PT158,TCY1, combinations thereof, and pharmaceutically acceptable saltsthereof, is variable in capsule/tablets/smart pills, resulting inintermittent, rather than constant dosing, the pills could be coded(e.g. by color or shape) to indicate which pills would be taken in whichorder (per part of a day, or per day or week or month of pill takingprotocol) to achieve the correct balance or GCR blockade (e.g. likedaily birth control pills with variable hormonal contents over thecourse of a month). In other words, some capsule/tablets/smart pillswould have just the opiate, some would have opiate plus one or severaldifferent doses of PT150 in the same capsule/tablet/smart pill, etc. Inexemplary embodiments, the patient would not be aware of the contents ofany given capsule/tablet/smart pill, but would know when in the courseof their dosing regimen they should take a particular coded version.

In exemplary embodiments, the smart capsules could identify bothquantity of pills taken as well as the kind of pill taken (with orwithout the ORG 34517, PT150, PT155, PT156, PT157, PT158, TCY1,combinations thereof, and pharmaceutically acceptable salts thereof) soas to provide a record for treating personnel as to what has been taken,but also to prevent opening of the smart pill if the wrong pill has beentaken out of its intended sequence.

The dose administered must be carefully adjusted according to age,weight and condition of the patient, as well as the route ofadministration, dosage form and regimen and the desired result. Thepharmaceutical compositions of the invention may be administered in thedosage forms in single or divided doses of one to four times daily. Itmay be advisable to start a patient on a low dose combination and workup gradually to a high dose combination.

Tablets of various sizes can be prepared, e.g., of about 1 to 2000 mg intotal weight, containing one or both of the active pharmaceuticalingredients, with the remainder being a physiologically acceptablecarrier of other materials according to accepted pharmaceuticalpractice.

These tablets can be scored to provide for fractional doses. Gelatincapsules can be similarly formulated.

Liquid formulations can also be prepared by dissolving or suspending oneor the combination of active substances in a conventional liquid vehicleacceptable for pharmaceutical administration so as to provide thedesired dosage in one to four teaspoonful.

Dosage forms can be administered to the patient on a regimen of, forexample, one, two, three, four, five, six, or other doses per day

In order to more finely regulate the dosage schedule, the activesubstances may be administered separately in individual dosage units atthe same time or carefully coordinated times. Since blood levels arebuilt up and maintained by a regulated schedule of administration, thesame result is achieved by the simultaneous presence of the twosubstances. The respective substances can be individually formulated inseparate unit dosage forms in a manner similar to that described above.In formulating the compositions, the active substances, in the amountsdescribed above, may be compounded according to accepted pharmaceuticalpractice with a physiologically acceptable vehicle, carrier, excipient,binder, preservative, stabilizer, flavor, etc., in the particular typeof unit dosage form.

Illustrative of the adjuvants which may be incorporated in tablets arethe following: a binder such as gum tragacanth, acacia, com starch orgelatin; an excipient such as dicalcium phosphate or cellulose; adisintegrating agent such as com starch, potato starch, alginic acid orthe like; a lubricant such as stearic acid or magnesium stearate; asweetening agent such as sucrose, aspartame, lactose or saccharin; aflavoring agent such as orange, peppermint, oil of wintergreen orcherry. When the dosage unit form is a capsule, it may contain inaddition to materials of the above type a liquid carrier such as a fattyoil. Various other materials may be present as coatings or to otherwisemodify the physical form of the dosage unit. For instance, tablets orcapsules may be coated with shellac, sugar or both. A syrup of elixirmay contain the active compound, water, alcohol or the like as thecarrier, glycerol as solubilizer, sucrose as sweetening agent, methyland propyl parabens as preservatives, a dye and a flavoring such ascherry or orange.

One embodiment of this invention includes methods of treating,preventing, or diagnosing a particular disease or condition byadministering the disclosed nanoparticles, composite nanoparticles,nanosuspension, or nanocapsules to a subject. In many instances, thenanoparticles, composite nanoparticles, or nanocapsules are administeredalone or can be included within a pharmaceutical composition. Aneffective amount of a pharmaceutical composition, generally, is definedas that amount sufficient to ameliorate, reduce, minimize, or limit theextent of the disease or condition. More rigorous definitions may apply,including elimination, eradication, or cure of the disease or condition.

“Nanoparticles” are solid particles of an average particle diameter of,for example, less than about 1 micron (micrometer). One micron is 1,000nanometers (nm). “Stabilized” nanoparticles are nanoparticles coatedwith a stabilizing material and having a reduced tendency foraggregation and loss of dispersion with respect to nanoparticles of thecompound of the invention without a stabilizing coating. A nano-spray isa spray containing nanoparticles or a spray that produces nanoparticles.A nanodispersion is a dispersion containing nanoparticles. Ananosuspension is a suspension containing nanoparticles. The liquidformulations useful herein may comprise a solvent, solution, suspension,microsuspension, nanosuspension, emulsion, microemulsion, gel or even amelt containing the active component or components.

In some embodiments the nanoparticles, nanofibers, or nanofibrils may bein the form of, or within or on, granules, powders, suspensions,solutions, dissolvable films, mats, webs, tablets, or releasable formsparticularly releasable dosage forms. Other particular useful forms areconcentrates to which a diluting liquid is added prior to use. Theproduct may also be sprayed onto the inner surface of a container towhich a liquid is added later prior to use and the nanoparticles,nanofibers, or nanofibrils, are released into the liquid.

Pharmaceutical compositions of the present invention can includenanoparticles, composite nanoparticles, nanosuspension, or nanocapsulesof the present invention. In certain non-limiting embodiments,pharmaceutical compositions may comprise, for example, at least about0.1% of an active ingredient or nanoparticles, composite nanoparticles,or nanocapsules, for example. In other embodiments, the an activeingredient or nanoparticles, composite nanoparticles, or nanocapsulesmay comprise between about 2% to about 75% of the weight of the unit, orbetween about 25% to about 60%, for example, and any range derivabletherein. In non-limiting examples of a derivable range from the numberslisted herein, a range of about 5 mg/kg/body weight to about 100mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered.

The composition may also include various antioxidants to retardoxidation of one or more active ingredient or nanoparticles, compositenanoparticles, nanosuspension, or nanocapsules. The prevention of theaction of microorganisms can be brought about by preservatives such asvarious antibacterial and antifungal agents, including but not limitedto parabens (e.g., methylparabens, propylparabens), chlorobutanol,phenol, sorbic acid, thimerosal or combinations thereof. In order toincrease the effectiveness of a treatment with the nanoparticles,nanogels, composite nanoparticles, nanosuspension, or nanocapsules ofthe present invention, it may be desirable to combine thesenanoparticles, composite nanoparticles, or nanocapsules with othertherapies effective in the treatment of a particular disease orcondition.

The formulations as described above may be administered for a prolongedperiod, that is, for as long as the potential for a disease or conditionremains or the symptoms continue.

Packaging/Treatment Kits

The present invention relates to a kit for conveniently and effectivelycarrying out the methods in accordance with the present invention. Suchkits may be suited for the delivery of solid oral forms such as tabletsor capsules. Such a kit may include a number of unit dosages. Such kitscan include a means for containing the dosages oriented in the order oftheir intended use. An example of a means for containing the dosages inthe order of their intended uses is a card. An example of such a kit isa “blister pack”. Blister packs are well known in the packaging industryand are widely used for packaging pharmaceutical unit dosage forms. Ifdesired, the blister can be in the form of a childproof blister, i.e., ablister that is difficult for a child to open, yet can be readily openedby an adult. If desired, a memory aid can be provided, for example inthe form of numbers, letters, or other markings or with a calendarfeature and/or calendar insert, designating the days and the sections ofa day in the treatment schedule in which the dosages can beadministered, such as an AM dose is packaged with a “mid day” and a PMdose.; or an AM dose is packaged with a PM dose. Alternatively, placebodosages, or vitamin or dietary supplements, either in a form similar toor distinct from the pharmaceutical active dosages, can be included.

In one aspect, the package, kit or container comprises a “blisterpackage” (also called a blister pack, or bubble pack). In one aspect,the blister package consists two or more separate compartments: Amdosage of this invention, and PM dosage of this invention, or mid-daydosage of this invention. This blister package is made up of twoseparate material elements: a transparent plastic cavity shaped to theproduct and its blister board backing. These two elements are thenjoined together with a heat sealing process which allows the product tobe hung or displayed. Exemplary types of “blister packages” include:Face seal blister packages, gang run blister packages, mock blisterpackages, interactive blister packages, slide blister packages.

Blister packs, clamshells or trays are forms of packaging used forgoods; thus, the invention provides for blister packs, clamshells ortrays comprising a composition (e.g., a (the multi-ingredientcombination of drugs of the invention) combination of activeingredients) of the invention. Blister packs, clamshells or trays can bedesigned to be non-reclosable, so consumers can tell if a package hasalready opened. They are used to package for sale goods where producttampering is a consideration, such as the pharmaceuticals of theinvention. In one aspect, a blister pack of the invention comprises amoulded PVC base, with raised areas (the “blisters”) to contain thetablets, pills, etc. comprising the combinations of the invention,covered by a foil laminate. Tablets, pills, etc. are removed from thepack either by peeling the foil back or by pushing the blister to forcethe tablet to break the foil. In one aspect, a specialized form of ablister pack is a strip pack.

In one aspect, a blister pack also comprises a method of packaging wherethe compositions comprising combinations of ingredients of the inventionare contained in-between a card and a clear PVC. The PVC can betransparent so the item (pill, tablet, geltab, etc.) can be seen andexamined easily; and in one aspect, can be vacuum-formed around a mouldso it can contain the item snugly and have room to be opened uponpurchase. In one aspect, the card is brightly colored and designeddepending on the item (pill, tablet, geltab, etc.) inside, and the PVCis affixed to the card using pre-formed tabs where the adhesive isplaced. The adhesive can be strong enough so that the pack may hang on apeg, but weak enough so that this way one can tear open the join andaccess the item. Sometimes with large items or multiple enclosed pills,tablets, geltabs, etc., the card has a perforated window for access. Inone aspect, more secure blister packs, e.g., for items such as pills,tablets, geltabs, etc. of the invention are used, and they can compriseof two vacuum-formed PVC sheets meshed together at the edges, with theinformative card inside.

In one aspect, blister packaging comprises at least two components(e.g., is a multi-ingredient combination of drugs of the invention): athermoformed “blister” which houses the product (e.g., a pharmaceuticalcombination of the invention), and then a “blister card” that is aprinted card with an adhesive coating on the front surface. During theassembly process, the blister component, which is most commonly made outof PVC, is attached to the blister card using a blister machine. Thismachine introduces heat to the flange area of the blister whichactivates the glue on the card in that specific area and ultimatelysecures the PVG blister to the printed blister card. The thermoformedPVG blister and the printed blister card can be as small or large.Conventional blister packs can also be sealed (e.g., using an AERGO 8DUO®, SCA Consumer Packaging, Inc., DeKalb, 111.) using regular heatseal tooling. This alternative aspect, using heat seal tooling, can sealcommon types of thermoformed packaging.

As discussed herein, the products of manufacture of the invention cancomprise the packaging of the therapeutic drug combinations of theinvention, alone or in combination, as “blister packages” or as aplurality of packettes, including as lidded blister packages, liddedblister or blister card or packets, or a shrink wrap.

In one aspect, laminated aluminum foil blister packs are used, e.g., forthe preparation of drugs designed to dissolve immediately in the mouthof a patient. This exemplary process comprises having the drugcombinations of the invention prepared as an aqueous solution(s) whichare dispensed (e.g., by measured dose) into an aluminum (e.g., alufoil)laminated tray portion of a blister pack. This tray is then freeze-driedto form tablets which take the shape of the blister pockets. The alufoillaminate of both the tray and lid fully protects any highly hygroscopicand/or sensitive individual doses. In one aspect, the pack incorporatesa child-proof peel open security laminate. In one aspect, the systemgive tablets an identification mark by embossing a design into thealufoil pocket that is taken up by the tablets when they change fromaqueous to solid state. In one aspect, individual ‘push-through’ blisterpacks/packettes are used, e.g., using hard temper aluminum (e.g.,alufoil) lidding material. In one aspect, hermetically-sealed highbarrier aluminum (e.g., alufoil) laminates are used. In one aspect, anyof the invention's products of manufacture, including kits or blisterpacks, use foil laminations and strip packs, stick packs, sachets andpouches, peelable and non-peelable laminations combining foil, paper,and film for high barrier packaging. Other means for containing saidunit dosages can include bottles and vials, wherein the bottle or vialcomprises a memory aid, such as a printed label for administering saidunit dosage or dosages. The label can also contain removable reminderstickers for placement on a calendar or dayminder to further help thepatient to remember when to take a dosage or when a dosage has beentaken.

Topical Formulations

The term “topical” as employed herein relates to the use of a compound,derivative or analogue as described herein, incorporated in a suitablepharmaceutical carrier, and applied at the site for exertion of localaction. Accordingly, such topical compositions including those forms inwhich the compound is applied externally by direct contact with the skinsurface to be treated. Conventional forms for this purpose includeointments, liniments, creams, shampoos, lotions, pastes, jellies,sprays, aerosols, soaps, and the like, and may be applied in patches orimpregnated dressings depending on the part of the body to be treated.The term “ointment” embraces formulations (including creams) havingoleaginous, absorption, water-soluble and emulsion-type bases, e.g.,petrolatum, lanolin, polyethylene glycols, as well as mixtures of these.

For topical use, the agent of the invention can be advantageouslyformulated using ointments, creams, liniments or patches as a carrier ofthe active ingredient. Also, these formulations may or may not containpreservatives, depending on the dispenser and nature of use. Suchpreservatives include those mentioned above, and methyl-, propyl-, orbutyl-parahydroxybenzoic acid, betain, chlorhexidine, benzalkoniumchloride, and the like. Various matrices for slow release delivery mayalso be used. Typically, the dose to be applied is in the range of about0.1 ng to about 100 mg per day, or about 1 ng to about 10 mg per day, orabout 10 ng to about 1 mg per day depending on the formulation.Non-limiting examples of topical products can include, withoutlimitation, application stick, mascara, eyebrow coloring products, eyeshadow or other eye lid coloring products, eyeliner, make-up removalproducts, antiaging products, facial or body powder, nail polish,mousse, sprays, styling gels, nail conditioner, bath and shower gels,shampoos, conditioners, cream rinses, hair dyes and coloring products,hair conditioners, sun tanning lotions and creams and sprays, sunscreensand sunblocks, skin conditioners, cold creams, moisturizers, hairsprays, soaps, body scrubs, exfoliants, astringents, depilatories andpermanent waving solutions, antidandruff formulations, antisweat andantiperspirant compositions, shaving, preshaving and after shavingproducts, moisturizers, deodorants, cold creams, cleansers, skin gels,and rinses.

Furthermore, the topical product can be applied topically through theuse of a patch or other delivery device. Delivery devices can include,but are not limited to, those that can be heated or cooled, as well asthose that utilize iontophoresis or ultrasound.

For instance, the topical product can be applied, for example, byapplying a composition in the form of a skin lotion, clear lotion, milkylotion, cream, gel, foam, ointment, paste, emulsion, spray, conditioner,tonic, cosmetic, application stick, pencil, foundation, nail polish,after-shave, or the like which is intended to be left on the skin orother keratinous tissue (i.e., a “leave-on” composition). After applyingthe composition to the keratinous tissue (e.g., skin), it in oneembodiment, it is left on for a period of at least about 15 minutes, orat least about 30 minutes, or at least about 1 hour, or for at leastseveral hours, e.g., up to about 12 hours. In one embodiment, thetopical product is left on overnight. In another embodiment, the topicalproduct is left on all day. Any part of the external portion of theface, hair, and/or nails can be treated, (e.g., face, lips, under-eyearea, eyelids, scalp, neck, torso, arms, legs, chest, hands, legs, feet,fingernails, toenails, scalp hair, eyelashes, eyebrows, etc.)

Any suitable method can be used to apply the topical product, includingbut not limited to for example using the palms of the hands and/orfingers or a device or implement (e.g., a cotton ball, swab, pad,applicator pen, spray applicator, eyebrow brush, eyebrow brush pencil,pencil, mascara brush, etc.) Another approach to ensure a continuousexposure of the keratinous tissue to at least a minimum level of thetopical product is to apply the compound by use of a patch applied,e.g., to the face. The patch can be occlusive, semi-occlusive ornonocclusive, and can be adhesive or non-adhesive. The topical productcan be contained within the patch or be applied to the skin prior toapplication of the patch. The patch can also include additional activessuch as chemical initiators for exothermic reactions such as thosedescribed in PCT application WO 9701313, and in U.S. Pat. Nos.5,821,250, 5,981,547, and 5,972,957 to Wu, et al. The patch can be lefton the for any suitable period of time. For example, a period of atleast about 5 minutes, or at least about 15 minutes, or at least about30 minutes, or at least about 1 hour, or at night as a form of nighttherapy, or in another embodiment all day.

Administration

The pharmaceutical compositions may be optimized for particular types ofdelivery. For example, pharmaceutical compositions for oral delivery areformulated using pharmaceutically acceptable carriers that are wellknown in the art. The carriers enable the agents in the composition tobe formulated, for example, as a tablet, pill, capsule, solution,suspension, sustained release formulation; powder, liquid or gel fororal ingestion by the subject.

The pharmaceutical compositions may also be delivered in an aerosolspray preparation from a pressurized pack, a nebulizer or from a drypowder inhaler. Suitable propellants that can be used in a nebulizerinclude, for example, dichlorodifluoro-methane, trichlorofluoromethane,dichlorotetrafluoroethane and carbon dioxide. The dosage can bedetermined by providing a valve to deliver a regulated amount of thecompound in the case of a pressurized aerosol. Compositions forinhalation or insufflation include solutions and suspensions inpharmaceutically acceptable, aqueous or organic solvents, or mixturesthereof, and powders. The liquid or solid compositions may containsuitable pharmaceutically acceptable excipients as set out above.Preferably the compositions are administered by the oral, intranasal orrespiratory route for local or systemic effect. Compositions inpreferably sterile pharmaceutically acceptable solvents may be nebulizedby use of inert gases. Nebulized solutions may be breathed directly fromthe nebulizing device or the nebulizing device may be attached to a facemask, tent or intermittent positive pressure breathing machine.Solution, suspension or powder compositions may be administered,preferably orally or nasally, from devices that deliver the formulationin an appropriate manner.

Typically, the composition may be applied repeatedly for a sustainedperiod of time topically on the part of the body to be treated, forexample, the eyelids, eyebrows, skin or scalp. The dosage regimen willgenerally involve regular, such as daily, administration for a period oftreatment of at least one month, or at least three months, or at leastsix months.

Alternatively, the composition may be applied intermittently, or in apulsed manner. Accordingly, an alternative embodiment of the inventionis to apply the composition on an intermittent or pulsed dosageschedule. For example, the composition of the invention may be used fortwo or more days, stopped, then restarted again at a time from between 2weeks to 3 months later, and at even more long-spaced intervals in thecase of the scalp.

The routes of administration of a compound of the present invention willvary, naturally, with the location and nature of the condition to betreated, and include, e.g., inhalation, intradermal, transdemial,parenteral, intravenous, intramuscular, intranasal, subcutaneous,percutaneous, intratracheal, intraperitoneal, intratumoral, perfusion,lavage, direct injection, and oral administration and formulation. Asdetailed below, GR antagonists may be administered as medical gases byinhalation or intubation, as injectable liquids by intravascular,intravenous, intra-arterial, intracerobroventicular, intraperitoneal,subcutaneous administration, as topical liquids or gels, or in solidoral dosage forms.

Moreover, the amounts may vary depending on the type of biologicalmatter (cell type, tissue type, organism genus and species, etc.) and/orits size (weight, surface area, etc.). It will generally be the casethat the larger the organism, the larger the dose. Therefore, aneffective amount for a mouse will generally be lower than an effectiveamount for a rat, which will generally be lower than an effective amountfor a dog, which will generally be lower than an effective amount for ahuman. The effective concentration of a compound of the presentinvention to achieve stasis, for example, in a human depends on thedosage form and route of administration. For inhalation, in someembodiments effective concentrations are in the range of 50 ppm to 500ppm, delivered continuously. For intravenous administration, in someembodiments effective concentrations are in the range of 0.5 to 50milligrams per kilogram of body weight delivered continuously.

Similarly, the length of time of administration may vary depending onthe type of biological matter (cell type, tissue type, organism genusand species, etc.) and/or its size (weight, surface area, etc.) and willdepend in part upon dosage form and route of administration. Inparticular embodiments, a compound of the present invention may beprovided for about or at least 30 seconds, 1 minute, 2 minutes, 3minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours,3 hours, four hours five hours, six hours, eight hours, twelve hours,twenty-four hours, or greater than twenty-four hours. A compound of thepresent invention may be administered in a single dos or multiple doses,with varying amounts of time between administered doses.

The treatments may include various “unit doses.” Unit dose is defined ascontaining a predetermined-quantity of the therapeutic composition. Thequantity to be administered, and the particular route and formulation,are within the skill of those in the clinical arts. A unit dose need notbe administered as a single injection but may comprise continuousinfusion over a set period of time. Alternatively, the amount specifiedmay be the amount administered as the average daily, average weekly, oraverage monthly dose.

In the case of transplant, the present invention may be used pre- and orpost-operatively to render host or graft materials quiescent. In aspecific embodiment, a 30 surgical site may be injected or perfused witha formulation comprising an GR antagonist. The perfusion may becontinued post-surgery, for example, by leaving a catheter implanted atthe site of the surgery.

Further Delivery Devices or Apparatuses

In some embodiments it is contemplated that methods or compositions willinvolve a specific delivery device or apparatus. Any method discussedherein can be implemented with any device for delivery or administrationincluding, but not limited, to those discussed herein.

For topical administration of GR antagonists of the invention may beformulated as solutions, gels, ointments, creams, suspensions, etc. asare well-known in the art. Systemic formulations may include thosedesigned for administration by injection or infusion, e.g.,subcutaneous, intravenous, intramuscular, intrathecal or intraperitonealinjection, as well as those designed for transdermal, transmucosal, oralor pulmonary administration.

The invention provides a topical pharmaceutical formulation for use intreatment of a subject, comprising the composition of the invention, andat least one pharmaceutically acceptable excipient.

The invention provides a method for treating a patient in need of suchtreatment comprising administration of the topical pharmaceuticalcomposition of the invention.

The invention provides a topical pharmaceutical formulation for use intreatment of a subject, comprising a composition of the invention, andat least one pharmaceutically acceptable excipient. The inventionfurther provides a topical formulation of the invention wherein saidformulation is in a form selected from the group consisting of: cream,lotion, gel, oil, ointment, suppository, spray, foam, liniment, aerosol,buccal and sublingual tablet or a transdermal device or patch forabsorption through the skin or mucous membranes. The invention furtherprovides a topical formulation of the invention wherein saidpharmaceutical formulation is a transdermal patch. The invention furtherprovides a topical formulation of the invention wherein saidpharmaceutical formulation is a buccal formulation.

For oral administration, the GR antagonists of the invention to beformulated as tablets, pills, dragees, capsules, liquids, gels, syrups,slurries, suspensions and the like, for oral ingestion by a patient tobe treated or oral liquid preparations such as, for example,suspensions, elixirs and solutions.

For buccal administration, the compositions may take the form oftablets, lozenges, etc. formulated in conventional manner. Otherintramucosal delivery might be by suppository or intranasally.

For administration directly to the lung by inhalation the compound ofinvention may be conveniently delivered to the lung by a number ofdifferent devices.

Another drug delivery system comprises one or more ball semiconductoraggregations and facilitating release of a drug stored in a reservoir.The first aggregate is used for sensing and memory, and a secondaggregation for control aspects, such as for pumping and dispensing ofthe drug. The system may communicate with a remote control system, oroperate independently on local power over a long period for delivery ofthe drug based upon a request of the patient, timed-release undercontrol by the system, or delivery in accordance with measured markers.PUMPS and Infusion Devices: An infusion pump or perfusor infuses fluids,medication or nutrients into a patient's circulatory system. Infusionpumps can administer fluids in very reliable and inexpensive ways. Forexample, they can administer as little as 0.1 mL per hour injections(too small for a drip), injections every minute, injections withrepeated boluses requested by the patient, up to maximum number per hour(e.g. in patient-controlled analgesia), or fluids whose volumes vary bythe time of day.

Implantable Drug Delivery System: Another drug delivery system comprisesone or more ball semiconductor aggregations and facilitating release ofa drug stored in a reservoir. The first aggregate is used for sensingand memory, and a second aggregation for control aspects, such as forpumping and dispensing of the drug. The system may communicate with aremote control system, or operate independently on local power over along period for delivery of the drug based upon a request of thepatient, timed-release under control by the system, or delivery inaccordance with measured markers.

Diagnostic Systems and Kits

A diagnostic kit may comprise some or all of the followingcomponents: 1) one or more standards comprised of one or more of thebiomarker(s) of the invention, such as cortisol; 2) a ligand, such as anantibody or a plurality of antibodies, that are specific for thebiomarker(s) that are to be assayed for using the kit; 3) writteninstructions; 4) diluents for samples and the standards; 5) a washbuffer; 6) color reagents; 7) stop solution; and 8) a ligand carrier,such as an antibody carrier, for example, a lateral flow device, or amicroplate with bound antibody, or polystyrene beads. An example of sucha kit is set forth in U.S. Patent Application Publication No.20120201747 (Altschul et al.), incorporated herein in its entirety.

While the invention has been described in detail and with reference tospecific examples thereof, it will be apparent to one skilled in the artthat various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.The invention will be illustrated in more detail with reference to thefollowing Examples, but it should be understood that the presentinvention is not deemed to be limited thereto.

EXAMPLES Example 1

Antiretroviral Activities of ORG34517 and PT155: Gain of Activity andDrop in Toxicity with PT155 in Comparison to ORG34517Preliminary results revealed that the thiosemicarbazone modification ofORG34517 in form of PT155 is beneficial to overall antiretroviralactivity and host cellular toxicity:

TABLE 12 Cytotoxicity and antiviral activity of ORG34517 and PT155versus human immunodeficiency type 1 strain LAI (HIV-1LAI) in mammaliancells. Cytotoxicity CC₅₀ (μM) and Anti-HIV-1_(LAI) cellular growth atfixed 100 μM activity EC₅₀ (μM)/ concentration (%, in parentheses) EC₉₀(μM) in PBM cells Drug PBM cells CCRF-CEM Vero EC₅₀ EC₉₀ SI₅₀ r² ORG20.6 15.4 44.3 8.9 21.6 2.3 0.96 34517 PT155 82.6 7.1 >100 5.5 16.2 15.00.93 (96.1) AZT* >100 14.3 56.0 PBM cells, primary human peripheralblood mononuclear cells. CCRF-CEM, human T-lymphoblastic acute T cellleukemia cells. Vero, African green monkey (grivet) Chlorocebus aethiops(syn. Cercopithecus aethiops) kidney epithelial cells. CC₅₀, cytotoxicconcentration 50%. EC₅₀, effective inhibitory concentration 50%. EC₉₀,effective inhibitory concentration 90%. SI₅₀, selectivity indexCC50/EC50. r2, coefficient of determination (r2 measure ofgoodness?+0of.fit) on EC₅₀ and EC₉₀. AZT, zidovudine(3′-azido-3′-deoxythymidine). *The given effective inhibitoryconcentrations (μM ± s.d.) for the positive control AZT were averagedand treated statistically from twenty (n = 20) independentdeterminations.

Method of Determination: HIV-1 Replication Reverse Transcriptase (RT)Assay

HIV-1LAI (=HIV-1BRU=LAV-1) was assayed in primary [freshly donated fromhealthy (tested HIV-1-negative, HBV-negative, and HCV-negative)_(—)blood donors, and isolated by single-step Ficoll-Hypaque centrifugationmethod] human peripheral blood mononuclear (PBM) cells in the presenceof a drug being evaluated. The parameter for antiviral activity wasreduction of RT activity in the cell supernatant after TritonX-100-mediated lysis of released virions, as measured by [5fÑ-3H]dTTP(5f{tilde over (R)}-tritiated thymidine 5′-triphosphate) incorporationinto poly(rA).poly(dT) directed by the primed RNA templatepoly(rA).oligo(dT). It should be noted that the assay did not detect RTinhibition by potential RT inhibitors per se, but indirectly quantifiedthe amount of released HIV-1 in the supernatant. The detailed assaymethodology was reported by Schinazi et al., as based on an older assaysystem of Spira et al. (ref. 38). The experiments were conducted intriplicate and treated statistically by regression curve analysis (r2coefficient of determination). The RT inhibitor AZT (zidovudine,3′-azido-3′-deoxythymidine; RETROVIR™) served as a positive control.Cytotoxicity on PBL cells exerted by the test compounds was determinedas described by Stuyver et al., by application of the CellTiter 96®AQueous One Solution Cell Proliferation Assay (Promega Corp., Madison,Wis.). Briefly, the phenazine ethosulfate (PES)-coupled reduction of thetetrazolium salt3-(4,5-dimethylthiazol-2-yl)-5-β-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) to a purple, water-soluble formazan by living, undamaged cells wasmeasured.

Example 2(11β,17β)-17-Hydroxy-11-[3,4-(methylenedioxy)phenyl]-17-(1-propyn-1-yl)estra-4,9-dien-3-one(ORG34517)×(2EZ)-2-{(11β,17β)-17-hydroxy-11-[3,4-(methylenedioxy)phenyl]-17-(1-propyn-1-yl)estra-4,9-dien-3-ylidene}hydrazinecarbothioamide[71.8% (E), 28.2% (Z)] hemihydrate×¾ acetone×ethanol (PT155)

Materials:

h ORG34517 [Pop Test Oncology LLC, Cliffside Park, N.J., USA;manufactured by Sai Life Sciences Ltd., Pune, India, Lot: SPO-MA1401-07;w (n1 n)=99.72% (HPLC, UV detection at 210 nm)]h Thiosemicarbazide puriss. p.a. [Sigma-Aldrich Corp., St. Louis, Mo.,USA, Lot: 1167177V (Fluka); w (m/m)=100.1% (iodometric titration), mp181° C. (dec.), residue on ignition <0.05%, metal trace analysis(inductively coupled plasma mass spectrometry)=50-5 mg/kg]h Glacial acetic acid (acetic acid 100% p.a.) [AppliChem GmbH,Darmstadt, Germany; Lot: 8Y002937; w (m/m)=100.0% (titration), water0.0% (Karl Fischer titration), acetic anhydride=0.05%, formicacid=0.01%, non-volatile matter=0.001%]

Instruction:

ORG34517 (M=430.54 g/mol, 2.000 g, 4.6453 mmol) and thiosemicarbazidepuriss. p.a. (M=91.14 g/mol, 435 mg, 4.7729 mmol) were suspended in 90%(v/v) aqueous ethanol (100 ml). Glacial acetic acid (1000μ1, 17.4854mmol) was added. The suspension was refluxed gently for 1 h. All solidswent into solution which turned light yellow during reflux.Afterwards, the yellow solution was left standing at room temperature(RT, f°=18.0° C.) for 12 min. Then the still warm yellow solution wasfiltered through one layer of filter paper. Residues were transferredand rinsed with 90% (v/v) aqueous ethanol (40 ml), and, successively,with water (40 ml). The yellow filtrate was cooled at +0-2° C. for 30min. After addition of water (40 ml), the precipitating suspension wasfrozen at −25° C. for 2 h. The evolved first yield of the cream yellow,crude PT155 was filtered and dried over CaCl2 in vacuo. The filtrate wasmixed with water (40 ml), and was frozen at −25° C. for 4 h. The evolvedsecond yield of the cream yellow, crude PT155 was filtered and driedover CaCl2 in vacuo. Both yields were combined.The crude product was dissolved in acetone (80 ml). Afterwards, water(2×40 ml) was added in portions under stirring to yield a yellowishemulsion. The precipitating emulsion with sticky yellow material pieceswas frozen at −25° C. for 4 h. The evolved first yield (1.677 g) of theyellow PT155 was filtered and dried over CaCl₂ in vacuo. The filtratewas mixed with water (20 ml) and was frozen at −25° C. for 1 h.Afterwards, water (20 ml) was added and the precipitating suspension wasfrozen at −25° C. for 1 h. The evolved second yield (139 mg) of theyellow PT155 was filtered and dried over CaCl₂ in vacuo. Both yieldswere combined (1.816 g).

-   Compound: PT155-   Molecular formula: ⅓ C₂₈H₃₀O₄×C₂₉H₃₃N₃O₃S×¾ C₃H₆O×⅛ C₂H₅OH×½ H₂O-   Molecular weight: 705.49 g/mol-   Yield: 1.816 g (74%)-   Elemental analysis: calculated: C, 69.52% H, 7.04% N, 5.96% S 4.55%    O, 12.95%    -   found: C, 69.81% H, 7.17% N, 5.82% S 4.25% O, 12.90% C 69.40% H,        7.13% N, 5.91% S 4.48% O, 12.70%.

¹H-NMR: (DMSO-d₆, ppm) 0.41 (3H, s; 18-CH₃, (E)-TSC*), 0.42 (1.18H, s;18-CH₃, (Z)-TSC**), 0.44 (1.393H, s; 18-CH₃, ORG34517), 1.06 (0.523H, t;³J (H, H)=7.1 Hz; ethanol CH₃), 1.23-2.77 (m; steroid CH and CH₂), 1.83(5.58H, br s; R—C≡C—CH₃ methyl, all three species), 2.09 (6.27H, s;acetone CH₃), 3.44 (0.348H, m; ethanol CH₂), 4.28 (0.393H, m; 11α-CH,(Z)-TSC), 4.30 (1H, d; ³J (H, H)=7.7 Hz; 11a-CH, (E)-TSC), 4.35 (0.174H,t; ³J (H, H)=5.1 Hz; ethanol OH), 4.38 (0.464H, d; ³J (H, H)=7.1 Hz;11a-CH, ORG34517), 5.11 (1H, s; 17β-OH, (E)-TSC), 5.12 (0.393H, s;17β-OH, (Z)-TSC), 5.14 (0.464H, s; 17β-OH, ORG34517), 5.66 (0.464H, s;4-CH, ORG34517), 5.86 (1H, s; 4-CH, (E)-TSC), 5.97 (3.716H, br s;O—CH₂—O benzodioxole, all three species), 5.97 (0.393H, s; 4-CH,(Z)-TSC), 6.60 (1.858H, d; ³J (H, H)=8.3 Hz; 5′-CH benzodioxole, allthree species), 6.67 (0.393H, s; 2′-CH benzodioxole, (Z)-TSC), 6.77(1.464H, s; 2′-CH benzodioxole, (E)-TSC and ORG34517), 6.78 (0.393H, d;³J (H, H)=8.0 Hz; 6′-CH benzodioxole, (Z)-TSC), 6.79 (1.464H, d; ³J (H,H)=8.0 Hz; 6′-CH benzodioxole, (E)-TSC and ORG34517), 7.51 (0.393H, brs; NH₂, H_(A), (Z)-TSC), 7.57 (1H, br s; NH₂, H_(A), (E)-TSC), 7.97(0.393H, br s; NH₂, H_(B), (Z)-TSC), 8.08 (1H, br s; NH₂, H_(B),(E)-TSC), 10.05 (1H, br s; N—H, (E)-TSC), 10.42 (0.393H, br s; N—H,(Z)-TSC). *,**(E or Z)-TSC=(E or Z)-thiosemicarbazone.

Example 31/17(11β,17β)-17-Hydroxy-11-[3,4-(methylenedioxy)phenyl]-17-(1-propyn-1-yl)estra-4,9-dien-3-one(ORG34517)×(2EZ)-2-{(11β,17β)-17-hydroxy-11-[3,4-(methylenedioxy)phenyl]-17-(1-propyn-1-yl)estra-4,9-dien-3-ylidene}-N-phenylhydrazinecarbothioamide[74.1% (E), 25.9% (Z)] 11/17 hydrate (PT156)

Materials: ORG34517

4-Phenylthiosemicarbazide 99% [ Sigma-Aldrich Corp., St. Louis, Mo.,USA, Lot: BCBL2527V (Aldrich); w (m/m)=99.7% (HClO₄ acidimetrictitration), w (n/n)=99.3% (HPLC, area %), mp 138-140° C.]Glacial acetic acid (acetic acid 100% p.a.) [AppliChem GmbH, Darmstadt,Germany; Lot: 8Y002937; w (m/m)=100.0% (titration), water 0.0% (KarlFischer titration), acetic anhydride≤0.05%, formic acid≤0.01%,non-volatile matter≤0.001%]

Instruction:

ORG34517 (M=430.54 g/mol, 4.000 g, 9.2907 mmol) and4-phenylthiosemicarbazide 99% (M=167.23 g/mol, 1.600 g, 9.5677 mmol)were suspended in 90% (v/v) aqueous ethanol (100 ml). Glacial aceticacid (1000μ1, 17.4854 mmol) was added. The suspension was refluxedgently for 1 h. All solids went into solution which turned deep yellowduring reflux.Afterwards, the yellow solution was left standing at room temperature(RT, l=15.6° C.) for 4 min. Then the still hot, cloudy, dirty yellowsolution was filtered through one layer of filter paper. Residues weretransferred and rinsed with 90% (v/v) aqueous ethanol (35 ml). The clearyellow filtrate was cooled at +0-2° C. for 4 h. The precipitatingsuspension was then frozen at −25° C. for 3 h. The evolved yield of thebright yellow, crude PT156 was filtered and dried over CaCl₂ in vacuo.The filtrate contained large amounts of impure ORG34517 and wasdiscarded.The crude product (3.463 g) was dissolved in acetone (30 ml).Afterwards, water (10 ml) was added in portions under stirring to yieldan oily yellow emulsion. The sticky emulsion was re-dissolved byaddition of acetone (15 ml). The mixture was then frozen at −25° C. for2 h. Afterwards, water (5 ml) was added which resulted in precipitationof sticky material pieces. The mixture was shaken vigorously for 30 s,and was frozen at −25° C. for 70 min. Finally, water (20 ml) was added,the mixture was shaken vigorously for 30 s, and the precipitatingemulsion was frozen at −25° C. for 75 min. The evolved first yield(3.285 g) of the bright yellow, crystalline PT156 was filtered and driedover CaCl₂ in vacuo. The filtrate was frozen at −25° C. for 12 h. Theevolved second yield (29 mg) of the bright yellow, crystalline PT156 wasfiltered and dried over CaCl₂ in vacuo. The filtrate contained smallamounts of pure ORG34517 and was discarded. Both yields were combined.

-   Compound: PT156-   Molecular weight: 616.73 g/mol-   Yield: 3.314 g (61%)-   Elemental analysis: calculated: C, 71.37% H, 6.55% N, 6.81% S 5.20%    O, 10.07%    -   found: C, 71.47% H, 6.83% N, 6.83% S 5.58% O, 10.05% C 71.18% H,        6.88% N, 6.87% S 5.45% O, 10.07%.

¹H-NMR: (DMSO-d₆, ppm) 0.42 (3H, s; 18-CH₃, (E)-PTSC*), 0.43 (1.05H, s;18-CH₃, (Z)-PTSC**), 0.44 (0.238H, s; 18-CH₃, ORG34517), 1.24-2.80 (m;steroid CH and CH₂), 1.83 (4.288H, br s; R—C≡C—CH₃ methyl, all threespecies), 2.09 (6H, s; acetone CH₃), 4.31 (0.35H, m; 11a-CH, (Z)-PTSC),4.33 (1H, d; ³J (H, H)=7.6 Hz; 11a-CH, (E)-PTSC), 4.37 (0.079H, d; ³J(H, H)=7.2 Hz; 11a-CH, ORG34517), 5.12 (1H, s; 17β-OH, (E)-PTSC), 5.13(0.35H, s; 17β-OH, (Z)-PTSC), 5.14 (0.079H, s; 17β-OH, ORG34517), 5.66(0.079H, s; 4-CH, ORG34517), 5.97 (2.859H, br s; O—CH₂—O benzodioxole,all three species), 6.00 (1.35H, s; 4-CH, (E)-PTSC and (Z)-PTSC), 6.62(1.429H, d; ³J (H, H)=8.0 Hz; 5′-CH benzodioxole, all three species),6.73 (0.35H, s; 2′-CH benzodioxole, (Z)-PTSC), 6.78 (1.079H, s; 2′-CHbenzodioxole, (E)-PTSC and ORG34517), 6.80 (0.35H, d; ³J (H, H)=8.0 Hz;6′-CH benzodioxole, (Z)-PTSC), 6.80 (1.079H, d; ³J (H, H)=8.0 Hz; 6′-CHbenzodioxole, (E)-PTSC and ORG34517), 7.14 (0.35H, m; 4″-H phenyl,(Z)-PTSC), 7.15 (1H, t; ³J (H, H)=7.4 Hz; 4″-H phenyl, (E)-PTSC), 7.31(0.7H, m; 3″,5″-H phenyl, (Z)-PTSC), 7.33 (2H, t; ³J (H, H)=8.0 Hz;3″,5″-H phenyl, (E)-PTSC), 7.60 (0.7H, d; ³J (H, H)=8.0 Hz; 2″,6″-Hphenyl, (Z)-PTSC), 7.62 (2H, d; ³J (H, H)=7.6 Hz; 2″,6″-H phenyl,(E)-PTSC), 9.83 (0.35H, br s; C₆H₅—N—H, (Z)-PTSC), 9.89 (1H, br s;C₆H₅—N—H, (E)-PTSC), 10.48 (1H, br s; ═N—N—H, (E)-PTSC), 10.83 (0.35H,br s; ═N—N—H, (Z)-PTSC). *,**(E or Z)-PTSC=(E orZ)-4-phenylthiosemicarbazone.

PT150 Purification of crude ORG34517 (PT150):(11β,17β)-17-hydroxy-11-[3,4-(methylenedioxy)phenyl]-17-(1-propyn-1-yl)estra-4,9-dien-3-one

Materials:

ORG34517 [Palisades Therapeutics, a division of Pop Test Oncology LLC,Cliffside Park, N.J., USA; manufactured by Wilmington PharmaTech CompanyLLC, Newark, Del., USA; delivered by TD2 Translational Drug Development,Scottsdale, Ariz., USA, Lot: 1930-120-23], CAS Registry Number:[189035-07-2] Doubly-distilled water, purified and sterile, Ethylacetate pro analysi [PanReac AppliChem GmbH, Lot: 0000518022; w(n/n)=99.9% (gas chromatography), w (H₂O) (m/m)=0.01% (Karl Fischertitration), ethanol<0.1%, methanol<0.02%, methyl acetate<0.02%, traceelements (Cr, Fe, Ni, Pb, Zn, P, S, K, Mg)<0.00001%, Si<0.00002%,Na<0.0002%, non-volatile matter<0.001%, acidity/alkalinity<0.0005 meq/g]Instruction: ORG34517 (M=430.54 g/mol, 4.000 g, 9.2907 mmol)

was suspended in distilled water (200 ml) in a 1000 ml round-bottomedflask. Then ethyl acetatepro analysi (EtOAc, 100 ml) wasadded as a supernatant. The flask was stoppered carefully, and themixture was shaken vigorously for exactly 1 min. After standing at roomtemperature (RT, 16.4° C.) for 1 min, the biphasic mixture was shakenvigorously for exactly 1 min. The initially yellow EtOAc phaselightened in color through the shaking procedures. After standing at RTfor 1 min, the mixture was transferred into a 500 ml separation funneland the phases were separated (ca. 10 min). The EtOAc phase wasisolated. The isolated aqueous phase was re-extracted with EtOAc (100ml) by shaking for exactly 10 s. The phases were separated (ca. 10 min).The first and the second EtOAc phase were combined. The isolatedresidual aqueous phase was re-extracted with EtOAc (50 ml) by shakingfor exactly 5 s. The phases were separated (ca. 20 min). All EtOAcphases were combined [the residual aqueous phase was cooled at +0-2° C.for 1 h, the evolved EtOAc phase was separated (ca. 20 min) and combinedwith the frozen EtOAc phase], and were frozen at −25° C. for 1.5 h. Theice-cold, ice-containing EtOAc phase was decanted from the frozenaqueous residues sticking at the glass surface into a 500 mlround-bottomed flask. The volume of the EtOAc solution was reduced atlow temperature (30-40° C.) in vacuo to a volume of ca. 20 ml whencrystalline masses appeared (the evaporation was stopped beforeevaporating to dryness!). The crystalline material sticking at the glasssurface was rinsed with 90% (v/v) aqueous ethanol (10 ml). Then themixture was frozen at −25° C. for 80 min. Afterwards, water (30 ml) wasadded and the crystallizing suspension was frozen at −25° C. for 15 min.The evolved first yield (2.076 g) of beautiful crystalline ORG34517(nearly white crystalline chucks) was filtered and dried over CaCl2 invacuo. The sticky residues in the flask. The volume of the EtOAcsolution was reduced at low temperature (30-40° C.) in vacuo to a volumeof ca. 20 ml when crystalline masses appeared (the evaporation wasstopped before evaporating to dryness!). The crystalline materialsticking at the glass surface was rinsed with 90% (v/v) aqueous ethanol(10 ml). Then the mixture was frozen at −25° C. for 80 min. Afterwards,water (30 ml) was added and the crystallizing suspension was frozen at−25° C. for 15 min. The evolved first yield (2.076 g) of beautifulcrystalline ORG34517 (nearly white crystalline chucks) was filtered anddried over CaCl2 in vacuo. The sticky residues in the flask were rinsedwith 90% (v/v) aqueous ethanol (5 ml) and combined with the firstfiltrate. A phase separation occurred. The separated EtOAc phase wasevaporated in the complete filtrate at the lowest possible temperaturein vacuo (ca. 30 min). The crystallizing suspension was frozen at −25°C. for 15 min. The evolved second yield (1.573 g) of ORG34517 wasfiltered and dried over CaCl2 in vacuo. Residues in the flask (and thevacuum flask) were rinsed with 90% (v/v) aqueous ethanol (4 ml), andwere combined with the second filtrate. The crystallizing suspension wasfrozen at −25° C. for 1.5 h. The evolved third yield (210 mg) ofORG34517 was filtered and dried over CaCl2 in vacuo. From the thirdfiltrate few substance (difference to 100%:141 mg) could be additionallyrecovered, if desired, by freezing for 1 h and treatment as before.

-   Compound: ORG34517 (PT150)-   Molecular formula: C₂₈H₃₀O₄-   Molecular weight: 430.54 g/mol-   Yield: 3.859 g (96%)-   Elemental analysis: calculated: C, 78.11% H, 7.02% O, 14.86%    -   found: C, 78.26% H, 7.04% O, 14.71% C, 78.44% H, 7.04% O,        14.71%.

¹H-NMR: (DMSO-d₆, ppm) 0.44 (3H, s; 18-CH₃), 1.24-2.76 (m; steroid CHand CH₂), 1.82 (3H, s; R—C≡C—CH₃ methyl), 4.37 (1H, d; ³J (H, H)=7.7 Hz;11

H), 5.14 (1H, s; 17

H), 5.66 (1H, s; 4-CH), 5.97 (2H, s; O—CH₂—O benzodioxole), 6.60 (1H, d;³J (H, H)=7.7 Hz; 5′-CH benzodioxole), 6.78 (1H, s; 2′-CH benzodioxole),6.79 (1H, d; ³J(H, H)=8.3 Hz; 6′-CH benzodioxole)

Second Step(11β,17β)-17-Hydroperoxy-11-[3,4-(methylenedioxy)phenyl]-17-(1-propyn-1-yl)estra-4,9-dien-3-one-(11β,17β)-17-hydroxy-11-[3,4-(methylenedioxy)phenyl]-17-(1-propyn-1-yl)estra-4,9-dien-3-one(1:1) (PT157)

Materials:

Crystalline ORG34517, purified as before (also capsule- ortablet-extracted amorphous material can be used), CAS Registry Number:[189035-07-2]Potassium ferricyanide K₃[Fe^(III)(CN)₆] [red prussiate of potash,potassium hexacyanoferrate(III)] BioChemica≥99% [AppliChem GmbH, Lot:6Y000987; w (m/m)=99.9% (titration), pH (1 M in H₂O) 6.1,chloride≤0.05%, sulfate≤0.005%, Pb≤0.002%]Sodium hydrogen carbonate (sodium bicarbonate) pro analysi NaHCO₃[AppliChem GmbH, Lot: 4W000829; w (m/m)=100.42% (titration), pH (5% inH₂O) 8.04 (20° C.), chloride≤0.001%, sulfate≤0.005%, phosphate≤0.005%,cations (K, Mg, Ca)≤0.005%, As<0.0001%, heavy metals (Cu, Fe,Pb)≤0.0005%]

Instruction:

Crystalline (also amorphous material can be used) ORG34517 (M=430.54g/mol, 3.136 g, 7.2839 mmol) was suspended in 90% (v/v) aqueous ethanol(110 ml) in a 500 ml round-bottomed flask. The suspension was heatedand, finally, refluxed until all solid had dissolved (ca. 10 min).Independently, a solution of potassium ferricyanide K₃[Fe^(III)(CN)₆](M=329.26 g/mol, 5.795 g, 17.6001 mmol=2.42×the molar amount ofORG34517) in water (65 ml) was prepared. The latter solution was pouredin one portion into the hot ORG34517 solution. Residues were transferredand rinsed with water (15 ml). The dark yellow solution was mixed with90% (v/v) aqueous ethanol (60 ml), and was refluxed for 8 min.Afterwards, a solution of sodium hydrogen carbonate (sodiumbicarbonate)pro analysi NaHCO₃ (M=84.01 g/mol, 1.497 g, 17.8193 mmol) inwater (15 ml) was added through the reflux condensor. Residues weretransferred and rinsed with water (8 ml). The solution initiallylightened in color, then darkened, and was refluxed for additional 12min. Then the dark yellow solution was cooled at room temperature (RT,l=17.1° C.) for 30 min. Crystals of K₃Na[Fe^(II)(CN)₆]×(H₂O)_(n)appeared. The crystallizing suspension was cooled at +0-2° C. for 2.5 h.Afterwards, the suspension was filtered through two layers of filterpaper. Residues were transferred and rinsed with 90% (v/v) aqueousethanol (50 ml). Finally, the wet K₃Na[Fe^(II)(CN)₆]×(H₂O)_(n) cake waspressed out with a spoon into the dark yellow filtrate (V 320 ml). Thisfiltrate was precipitated by addition of water (65 ml). Theprecipitating suspension was frozen at −25° C. for 1 h. Then additionalwater (35 ml) was added. The precipitating suspension was frozen at −25°C. for 4.5 h. The evolved first yield (2.464 g) of beige (off-white)PT157 was filtered on a sintered glass filter. The material was filtereddry on the filter by suction for 30 min. Successively, the material waswashed on the filter with water (200 ml) free of yellow iron compounds,and was dried over CaCl₂ in vacuo. The filtrate was transferred andrinsed with water (100 ml), and was frozen at −25° C. for 3.5 h. Theevolved second yield (304 mg) of beige (off-white) PT157 was filtered ona sintered glass filter. The material was filtered dry on the filter bysuction for 30 min. Successively, the material was washed on the filterwith water (200 ml) free of yellow iron compounds, and was dried overCaCl₂ in vacuo. Both yields were combined. The dark green filtrate wasdiscarded.

-   Compound: PT157-   Molecular formula: C₅₆E1₆₀O₉-   Molecular weight: 877.07 g/mol-   Yield: 2.768 g (87%)-   Elemental analysis: calculated: C, 76.69% H, 6.90% N, 0.00% O,    16.42%    -   found: C, 76.85% H, 7.04% N, 0.30% O, 15.09% C 76.98% H, 7.12%        N, 0.31% O, 15.27%-   Purity: from the nitrogen content (0.30%), caused by admixture of    K₃Na[Fe^(II)(CN)₆] (which has a nitrogen content of N 23.86%), it    could be calculated that PT157 has a purity of 98.74% (m/m) [or    96.93% (n/n)]

¹H-NMR: (DMSO-d₆, ppm) 0.44 (3H, s; 18-CH₃), 1.24-2.76 (m; steroid CHand CH₂), 1.83 (3H, s; R—C≡C—CH₃ methyl), 4.38 (1H, d; ³J(H, H)=7.1 Hz;11

H), 5.14 (1H, s; 17

H), 5.66 (1H, s; 4-CH), 5.97 (2H, s; O—CH₂—O benzodioxole), 6.61 (1H, d;³J(H, H)=8.3 Hz; 5′-CH benzodioxole), 6.78 (1H, s; 2′-CH benzodioxole),6.79 (1H, d; ³J (H, H)=8.3 Hz; 6′-CH benzodioxole)

Example 4

Virus Screened: Japanese encepahalitis virus Virus Strain: SA-14 CellLine: Vero 76 Vehicle: DMSO Drug Conc. Range: 0.1-100 μg/ml ControlConc. Range: 0.00001-0.01 μg/ml Experiment Number JEV-043

Control Control Assay Drug Name Order Control Assay Name EC₅₀ EC₉₀ CC₅₀SI₅₀ SI₉₀ Infergen Primary Visual (Cytopathic 0.000042 >0.01 >238effect/Toxicity) Infergen Primary Neutral Red (Cytopathic0.000038 >0.01 >263 effect/Toxicity) EC₅₀-compound concentration thatreduces viral replication by 50% SI₅₀ − CC₅₀/EC₅₀ EC₉₀-compoundconcentration that reduces viral replication by 90% SI₉₀ − CC₅₀/EC₉₀CC₅₀-compound concentration that reduces cell viability by 50%

Screening Results

Date Drug Received Compound Assay ARB No. m/dd/yy Name/ID Order DrugAssay Name EC₅₀ EC₉₀ CC₅₀ SI₅₀ SI₉₀ 16-000163 May 27, 2016 PT155Secondary Visual (Cytopathic 10 >100 >10 effect/Toxicity) 16-000163 May27, 2016 PT155 Secondary Neutral Red 2.9 >100 >34 (Cytopathiceffect/Toxicity)

Example 5

Virus Screened: West Nile virus Virus Strain: Kern 515, WN02 Cell Line:Vero 76 Vehicle: DMSO Drug Conc. Range: 0.1-100 μg/ml Control Conc.Range: 0.00001-0.01 μg/ml Experiment Number: WNV-0870

Control Control Drug Assay Name Order Control Assay Name EC₅₀ EC₉₀ CC₅₀SI₅₀ SI₉₀ Infergen Primary Visual 0.00001 >0.01 >1000 (Cytopathiceffect/Toxicity) Infergen Primary Neutral Red 0.000056 >0.01 >180(Cytopathic effect/Toxicity) EC₅₀-compound concentration that reducesviral replication by 50% EC₉₀-compound concentration that reduces viralreplication by 90% CC₅₀-compound concentration that reduces cellviability by 50% SI₅₀-CC₅₀/EC₅₀ SI₉₀-CC₅₀/EC₉₀

Screening Results

Date Drug Received Compound Assay ARB No. m/dd/yy Name/ID Order DrugAssay Name EC₅₀ EC₉₀ CC₅₀ SI₅₀ SI₉₀ 16-000163 May 26, 2016 PT155 PrimaryVisual (Cytopathic >22 22 0 effect/Toxicity) 16-000163 May 26, 2016PT155 Primary Neutral Red (Cytopathic >15 15 0 effect/Toxicity)

Example 6

Virus Screened: Yellow fever virus Virus Strain: 17D Cell Line: Vero 76Vehicle: DMSO Drug Conc. Range: 0.1-100 μg/ml Control Conc. Range:0.1-100 μg/ml Experiment Number: YFV-1177

Control Control Drug Assay Control Assay Name Order Name EC₅₀ EC₉₀ CC₅₀SI₅₀ SI₉₀ 6- Primary Visual 0.28 >100 >357 Azauridine (Cytopathiceffect/ Toxicity) 6- Primary Neutral Red 0.67 34 51 Azauridine(Cytopathic effect/ Toxicity) EC₅₀-compound concentration that reducesviral replication by 50% EC₉₀-compound concentration that reduces viralreplication by 90% CC₅₀-compound concentration that reduces cellviability by 50% SI₅₀-CC₅₀/EC₅₀ SI₉₀-CC₅₀/EC₉₀

Screening Results

Date Drug Received Compound Assay ARB No. m/dd/yy Name/ID Order DrugAssay Name EC₅₀ EC₉₀ CC₅₀ SI₅₀ SI₉₀ 16-000163 May 26, 2016 PT155 PrimaryVisual (Cytopathic 32 32 1 effect/Toxicity) 16-000163 May 26, 2016 PT155Primary Neutral Red (Cytopathic >14 14 0 effect/Toxicity)

Example 7

Virus Screened: Enterovirus 71 Virus Strain: Tainan/4643/98 Cell Line:Vero 76 Vehicle: DMSO Drug Conc. Range: 0.1-100 μg/ml Control Conc.Range: 0.01-10 μg/ml Experiment Number: ENTV-057

Control Control Drug Assay Name Order Control Assay Name EC₅₀ EC₉₀ CC₅₀SI₅₀ SI₉₀ Pirodavir Secondary Visual 0.23 >10 >43 (Cytopathiceffect/Toxicity) Pirodavir Secondary Neutral Red 0.081 >10 >120(Cytopathic effect/Toxicity) EC₅₀-compound concentration that reducesviral replication by 50% EC₉₀-compound concentration that reduces viralreplication by 90% CC₅₀-compound concentration that reduces cellviability by 50% SI₅₀-CC₅₀/EC₅₀ SI₉₀-CC₅₀/EC₉₀

Screening Results

Date Drug Received Compound Assay ARB No. m/dd/yy Name/ID Order DrugAssay Name EC₅₀ EC₉₀ CC₅₀ SI₅₀ SI₉₀ 16-000163 May 27, 2016 PT155Secondary Visual (Virus yield >100 >100 0 reduction)/Neutral Red(Toxicity) 16-000163 May 27, 2016 PT155 Secondary Neutral Red1.7 >100 >59 (Cytopathic effect/Toxicity)

Example 8

Virus Screened: Poliovirus 3 Virus Strain: WM-3 Cell Line: Vero 76Vehicle: DMSO Drug Conc. Range: 0.032-100 μg/ml Control Conc. Range:0.0032-10 μg/ml Experiment Number: POV-0099

Control Control Drug Assay Control Assay Name Order Name EC₅₀ EC₉₀ CC₅₀SI₅₀ SI₉₀ Pirodavir Secondary Visual 0.14 7.8 56 (Cytopathic effect/Toxicity) Pirodavir Secondary Neutral Red 0.058 7.8 130 (Cytopathiceffect/ Toxicity) EC₅₀-compound concentration that reduces viralreplication by 50% EC₉₀-compound concentration that reduces viralreplication by 90% CC₅₀-compound concentration that reduces cellviability by 50% SI₅₀-CC₅₀/EC₅₀ SI₉₀-CC₅₀/EC₉₀

Screening Results

Date Drug Received Compound Assay ARB No. m/dd/yy Name/ID Order DrugAssay Name EC₅₀ EC₉₀ CC₅₀ SI₅₀ SI₉₀ 16-000163 May 27, 2016 PT155Secondary Visual (Virus yield 10 >100 >10 reduction)/Neutral Red(Toxicity) 16-000163 May 27, 2016 PT155 Secondary Neutral Red2.9 >100 >34 (Cytopathic effect/Toxicity)

Example 9

Virus Screened: Zika virus Virus Strain: MR766 Cell Line: HUH 7 Vehicle:DMSO Drug Conc. Range: 0.1-100 μg/ml Control Conc. Range: 0.1-100 μg/mlExperiment Number: Zika-073

Control Control Drug Assay Name Order Control Assay Name EC₅₀ EC₉₀ CC₅₀SI₅₀ SI₉₀ 6- Secondary Visual (Virus yield reduction)/ 0.89 >100 >112Azauridine Neutral Red (Toxicity) 6- Secondary Neutral Red 4.1 >100 >24Azauridine (Cytopathic effect/Toxicity) EC₅₀-compound concentration thatreduces viral replication by 50% EC₉₀-compound concentration thatreduces viral replication by 90% CC₅₀-compound concentration thatreduces cell viability by 50% SI₅₀-CC₅₀/EC₅₀ SI₉₀-CC₅₀/EC₉₀

Screening Results

Date Drug Received Compound Assay ARB No. m/dd/yy Name/ID Order DrugAssay Name EC₅₀ EC₉₀ CC₅₀ SI₅₀ SI₉₀ 16-000162 May 18, 2016 PT150Secondary Visual (Virus yield 1.27 4.3 3.4 reduction)/Neutral Red(Toxicity) 16-000162 May 18, 2016 PT150 Secondary Neutral Red 1.6 4.32.7 (Cytopathic effect/Toxicity) 16-000163 May 18, 2016 PT155 SecondaryVisual (Virus yield 0.82 >100 >122 reduction)/Neutral Red (Toxicity)16-000163 May 18, 2016 PT155 Secondary Neutral Red 0.056 >100 >1800(Cytopathic effect/Toxicity)

Example 10

Virus Screened: NV Virus Strain: GT1 Cell Line: HG23 Vehicle: DMSO DrugConc. Range: 0.1-100 uM Control Conc. Range: 3.7-100 uM ExperimentNumber: 16-15

Control Control Drug Assay Name Order Control Assay Name EC₅₀ EC₉₀ CC₅₀SI₅₀ SI₉₀ 2′ C-methyl primary RNA Hybridization (Replicon)/ 6.419 >300 >47 >16 cytidine RNA Hybridization (Toxicity) 16-000162 primaryRNA Hybridization (Replicon)/ 1.0 9.3 >100 >100 >11 (Mar. 17, 2016) RNAHybridization (Toxicity) 16-000163 primary RNA Hybridization (Replicon)/42 115 >100 >2 >1 (Mar. 17, 2016) RNA Hybridization (Toxicity) 16-000164primary RNA Hybridization (Replicon)/ >100 >100 >100 1 1 (Mar. 17, 2016)RNA Hybridization (Toxicity) EC₅₀-compound concentration that reducesviral replication by 50% EC₉₀-compound concentration that reduces viralreplication by 90% CC₅₀-compound concentration that reduces cellviability by 50% SI₅₀-CC₅₀/EC₅₀ SI₉₀-CC₅₀/EC₉₀

Screening Results

Date Drug Received Compound Assay ARB No. m/dd/yy Name/ID Order DrugAssay Name EC₅₀ EC₉₀ CC₅₀ SI₅₀ SI₉₀ 16-000162 May 9, 2016 PT150 primaryRNA Hybridization 1.2 8.1 >100 >83 >12 (Replicon)/RNA Hybridization(Toxicity) 16-000163 May 9,2016 PT155 primary RNA Hybridization 37141.0 >100 >3 >1 (Replicon)/RNA Hybridization (Toxicity) 16-000164 May9, 2016 PT156 primary RNA Hybridization >100 >100 >100 1 1(Replicon)/RNA Hybridization (Toxicity)

Example 11

Virus Screened: MNV Virus Strain: MNV-1 Cell Line: RAW267.4 Vehicle:DMSO Drug Conc. Range: 0.03-100 uM Control Conc. Range: 1.0-30 uMExperiment Number: 16-14

Control Assay Control Assay Control Drug Name Order Name EC₅₀ EC₉₀ CC₅₀SI₅₀ SI₉₀ 2′ C-methyl primary Neutral Red 1.0 3.1 >30 >30 >10 cytidine(Cytopathology)/ Neutral Red (Toxicity) 16-000162 primary Neutral Red0.086 0.273 33 384 121 (Mar. 17, 2016) (Cytopathology)/ Neutral Red(Toxicity) 16-000163 primary Neutral Red 0.082 0.267 35 427 131 (Mar.17, 2016) (Cytopathology)/ Neutral Red (Toxicity) 16-000164 primaryNeutral Red 0.091 0.29 33 363 114 (Mar. 17, 2016) (Cytopathology)/Neutral Red (Toxicity) EC₅₀-compound concentration that reduces viralreplication by 50% EC₉₀-compound concentration that reduces viralreplication by 90% CC₅₀-compound concentration that reduces cellviability by 50% SI₅₀-CC₅₀/EC₅₀ SI₉₀-CC₅₀/EC₉₀

Screening Results

Date Drug Received Compound Assay ARB No. m/dd/yy Name/ID Order DrugAssay Name EC₅₀ EC₉₀ CC₅₀ SI₅₀ SI₉₀ 16-000162 May 9, 2016 PT150 primaryNeutral Red 0.097 0.309 34 351 110 (Cytopathology)/ Neutral Red(Toxicity) 16-000163 May 9, 2016 PT155 primary Neutral Red 0.087 0.29134 391 117 (Cytopathology)/ Neutral Red (Toxicity) 16-000164 May 9, 2016PT156 primary Neutral Red 0.096 0.304 33 344 109 (Cytopathology)/Neutral Red (Toxicity)

While the invention has been described in detail and with reference tospecific examples thereof, it will be apparent to one skilled in the artthat various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

What is claimed is:
 1. A method of treating and/or preventing a viralcondition in a patient comprising: selecting a patient in need oftreating and/or preventing a viral condition; administering to thepatient at least one active agent selected from the group consisting ofPT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof; administering at least oneadditional active agent selected from the group consisting of othermolecules with potential to bind viral glucocorticoid response elements(GREs), retinazone, RU486, derivatives thereof, wherein the viralcondition is to prevent or eliminate acute viral infection, to diminishintensity of viral infection, to diminish length of viral infection, tospeed time to resolution and healing of viral infection, to speed timeto suppression of viral infection, to increase likelihood of viraleradication, and/or to diminish infectivity of viral infection, withHepatitis C virus, Bovine Viral Diarrhea virus, Ebola-like viruses,Hepatitis B virus, Mouse mammary tumor virus, Human ImmunodeficiencyVirus-1 (HIV-1), Varicella-Zoster virus (chicken pox; VZV),Cytomegalovirus (CMV), Human Herpes Virus-6 (HHV-6), Human HerpesVirus-7 (HHV-7), Kaposi's Sarcoma-Associated Herpes virus (or HumanHerpes Virus-8; HHV-8), Variola (Small Pox) virus, Vaccinia virus,Cowpox virus, Monkeypox virus.
 2. A method of treating and/or preventinga viral condition in a patient comprising: selecting the patient in needof treating and/or preventing a viral condition; administering at leastone active agent selected from the group consisting of PT150, PT155,PT156, PT157, PT158, TCY1, combinations thereof, and pharmaceuticallyacceptable salts thereof; administering at least one additional activeagent selected from the group consisting of molecules with potential tobind viral glucocorticoid response elements (GREs), retinazone, RU486,and derivatives thereof, wherein the viral condition is to prevent acuteviral infection from becoming chronic active or latent infection withHepatitis C virus, Bovine Viral Diarrhea virus, Ebola-like viruses,Hepatitis B virus, Mouse mammary tumor virus, Human ImmunodeficiencyVirus-1 (HIV-1), Varicella-Zoster virus (chicken pox; VZV),Cytomegalovirus (CMV), Human Herpes Virus-6 (HHV-6), Human HerpesVirus-7 (HHV-7), Kaposi's Sarcoma-Associated Herpes virus (or HumanHerpes Virus-8; HHV-8), Variola (Small Pox) virus, Vaccinia virus,Cowpox virus, Monkeypox virus.
 3. A method of treating and/or preventinga viral condition in a patient comprising: selecting a patient in needof treating and/or preventing a viral condition; administering at leastone active agent selected from the group consisting of PT150, PT155,PT156, PT157, PT158, TCY1, combinations thereof, and pharmaceuticallyacceptable salts thereof; administering at least one additional activeagent selected from the group consisting of molecules with potential tobind viral glucocorticoid response elements (GREs), retinazone, RU486,or their derivatives, wherein the viral condition is to prevent chroniclatent viral infection from becoming active (reactivation), to diminishintensity of viral reactivation, to diminish length of viralreactivation, to speed time to resolution and healing of viralreactivation, to speed time to suppression of viral reactivation, toincrease likelihood of viral eradication, and/or to diminish infectivityof viral reactivation with: Hepatitis C virus, Bovine Viral Diarrheavirus, Ebola-like viruses, Hepatitis B virus, Mouse mammary tumor virus,Human Immunodeficiency Virus-1 (HIV-1), Varicella-Zoster virus (chickenpox; VZV), Cytomegalovirus (CMV), Human Herpes Virus-6 (HHV-6), HumanHerpes Virus-7 (HHV-7), Kaposi's Sarcoma-Associated Herpes virus (orHuman Herpes Virus-8; HHV-8), Variola (Small Pox) virus, Vaccinia virus,Cowpox virus, Monkeypox virus.
 4. A method of treating and/or preventinga viral condition in a patient comprising: selecting a patient in needof treating and/or preventing a viral condition; administering at leastone active agent selected from the group consisting of PT150, PT155,PT156, PT157, PT158, TCY1, combinations thereof, and pharmaceuticallyacceptable salts thereof; administering at least one additional activeagent selected from the group consisting of molecules with potential tobind viral glucocorticoid response elements (GREs), retinazone, RU486 ortheir derivatives wherein the viral condition is to inactivate latentpro-viral genome eliminating (“curing”) chronic viral infections withHepatitis C virus, Bovine Viral Diarrhea virus, Ebola-like viruses,Hepatitis B virus, Mouse mammary tumor virus, Human ImmunodeficiencyVirus-1 (HIV-1), Varicella-Zoster virus (chicken pox; VZV),Cytomegalovirus (CMV), Human Herpes Virus-6 (HHV-6), Human HerpesVirus-7 (HHV-7), Kaposi's Sarcoma-Associated Herpes virus (or HumanHerpes Virus-8; HHV-8), Variola (Small Pox) virus, Vaccinia virus,Cowpox virus, Monkeypox virus.
 5. A method of treating and/or preventinga viral condition in a patient comprising: selecting a patient in needof treating and/or preventing a viral condition; administering an activeagent selected from the group consisting of PT150, PT155, PT156, PT157,PT158, TCY1, combinations thereof, and pharmaceutically acceptable saltsthereof; administering at least one additional active agent selectedfrom the group consisting of molecules with potential to bind viral PS,annexin-5, anti-PS monoclonal or polyclonal antibodies, bavituximab,wherein the viral condition is to prevent or eliminate acute viralinfection, to diminish intensity of viral infection, to diminish lengthof viral infection, to speed time to resolution and healing of viralinfection, to speed time to suppression of viral infection, to increaselikelihood of viral eradication, and/or to diminish infectivity of viralinfection.
 6. The method of claim 5, wherein the viral condition isselected from the group consisting of Ebola and Marburg virus(Filoviridae); Ross River virus, chikungunya virus, Sindbis virus,eastern equine encephalitis virus (Togaviridae, Alphavirus), vesicularstomatitis virus (Rhabdoviridae, Vesiculovirus), Amapari virus, Pichindévirus, Tacaribe virus, Junin virus, Machupo virus (Arenaviridae,Mammarenavirus), West Nile virus, dengue virus, yellow fever virus(Flaviviridae, Flavivirus); human immunodeficiency virus type 1(Retroviridae, Lentivirus); Moloney murine leukemia virus (Retroviridae,Gammaretrovirus); influenza A virus (Orthomyxoviridae); respiratorysyncytial virus (Paramyxoviridae, Pneumovirinae, Pneumovirus); vacciniavirus (Poxviridae, Chordopoxvirinae, Orthopoxvirus); herpes simplexvirus type 1, herpes simplex virus type 2 (Herpesviridae,Alphaherpesvirinae, Simplexvirus); human cytomegalovirus (Herpesviridae,Betaherpesvirinae, Cytomegalovirus); Autographa californicanucleopolyhedrovirus (Baculoviridae, Alphabaculoviridae) (an insectvirus); Ebola and Marburg virus (Filoviridae); Semliki Forest virus,Ross River virus, chikungunya virus, O'nyong-nyong virus, Sindbis virus,eastern/western/Venezuelan equine encephalitis virus (Togaviridae,Alphavirus); rubella (German measles) virus (Togaviridae, Rubivirus);rabies virus, Lagos bat virus, Mokola virus (Rhabdoviridae, Lyssavirus);Amapari virus, Pichindé virus, Tacaribe virus, Junin virus, Machupovirus, Guanarito virus, Sabia virus, Lassa virus (Arenaviridae,Mammarenavirus); West Nile virus, dengue virus, yellow fever virus, Zikavirus, Japanese encephalitis virus, St. Louis encephalitis virus,tick-borne encephalitis virus, Omsk hemorrhagic fever virus, KyasanurForest virus (Flaviviridae, Flavivirus); human hepatitis C virus(Flaviviridae, Hepacivirus); human immunodeficiency virus type 1(Retroviridae, Lentivirus); influenza A/B virus (Orthomyxoviridae, thecommon ‘flu’ virus); respiratory syncytial virus (Paramyxoviridae,Pneumovirinae, Pneumovirus); Hendra virus, Nipah virus (Paramyxoviridae,Paramyxovirinae, Henipavirus); measles virus (Paramyxoviridae,Paramyxovirinae, Morbillivirus); variola major (smallpox) virus(Poxviridae, Chordopoxvirinae, Orthopoxvirus); human hepatitis B virus(Hepadnaviridae, Orthohepadnavirus); hepatitis delta virus (hepatitis Dvirus) (unassigned Family, Deltavirus); herpes simplex virus type 1,herpes simplex virus type 2 (Herpesviridae, Alphaherpesvirinae,Simplexvirus); human cytomegalovirus (Herpesviridae, Betaherpesvirinae,Cytomegalovirus).
 7. A method of treating and/or preventing a viralcondition in a patient comprising: selecting a patient in need oftreating and/or preventing a viral condition; administering to thepatient at least one active agent selected from the group consisting ofPT150, PT155, PT156, PT157, PT158, TCY1, combinations thereof, andpharmaceutically acceptable salts thereof; administering at least oneadditional active agent selected from the group consisting of moleculeswith potential to bind viral PS, annexin-5, anti-PS monoclonal orpolyclonal antibodies, bavituximab, wherein the viral condition is toprevent acute viral infection from becoming chronic active or latentinfection.
 8. The method of claim 7, wherein the viral condition isselected from the group consisting of Ebola and Marburg virus(Filoviridae); Ross River virus, chikungunya virus, Sindbis virus,eastern equine encephalitis virus (Togaviridae, Alphavirus), vesicularstomatitis virus (Rhabdoviridae, Vesiculovirus), Amapari virus, Pichindévirus, Tacaribe virus, Junin virus, Machupo virus (Arenaviridae,Mammarenavirus), West Nile virus, dengue virus, yellow fever virus(Flaviviridae, Flavivirus); human immunodeficiency virus type 1(Retroviridae, Lentivirus); Moloney murine leukemia virus (Retroviridae,Gammaretrovirus); influenza A virus (Orthomyxoviridae); respiratorysyncytial virus (Paramyxoviridae, Pneumovirinae, Pneumovirus); vacciniavirus (Poxviridae, Chordopoxvirinae, Orthopoxvirus); herpes simplexvirus type 1, herpes simplex virus type 2 (Herpesviridae,Alphaherpesvirinae, Simplexvirus); human cytomegalovirus (Herpesviridae,Betaherpesvirinae, Cytomegalovirus); Autographa californicanucleopolyhedrovirus (Baculoviridae, Alphabaculoviridae) (an insectvirus); Ebola and Marburg virus (Filoviridae); Semliki Forest virus,Ross River virus, chikungunya virus, O'nyong-nyong virus, Sindbis virus,eastern/western/Venezuelan equine encephalitis virus (Togaviridae,Alphavirus); rubella (German measles) virus (Togaviridae, Rubivirus);rabies virus, Lagos bat virus, Mokola virus (Rhabdoviridae, Lyssavirus);Amapari virus, Pichindé virus, Tacaribe virus, Junin virus, Machupovirus, Guanarito virus, Sabia virus, Lassa virus (Arenaviridae,Mammarenavirus); West Nile virus, dengue virus, yellow fever virus, Zikavirus, Japanese encephalitis virus, St. Louis encephalitis virus,tick-borne encephalitis virus, Omsk hemorrhagic fever virus, KyasanurForest virus (Flaviviridae, Flavivirus); human hepatitis C virus(Flaviviridae, Hepacivirus); human immunodeficiency virus type 1(Retroviridae, Lentivirus); influenza A/B virus (Orthomyxoviridae, thecommon ‘flu’ virus); respiratory syncytial virus (Paramyxoviridae,Pneumovirinae, Pneumovirus); Hendra virus, Nipah virus (Paramyxoviridae,Paramyxovirinae, Henipavirus); measles virus (Paramyxoviridae,Paramyxovirinae, Morbillivirus); variola major (smallpox) virus(Poxviridae, Chordopoxvirinae, Orthopoxvirus); human hepatitis B virus(Hepadnaviridae, Orthohepadnavirus); hepatitis delta virus (hepatitis Dvirus) (unassigned Family, Deltavirus); herpes simplex virus type 1,herpes simplex virus type 2 (Herpesviridae, Alphaherpesvirinae,Simplexvirus); human cytomegalovirus (Herpesviridae, Betaherpesvirinae,Cytomegalovirus).